Serum Bactericidal Activity Assay Research Articles

Natural Brucella melitensis Infection and Rev. 1 Vaccination Induce Specific Brucella O-Polysaccharide Antibodies Involved in Complement Mediated Brucella Cell Killing.

Vaccination against brucellosis using live attenuated strains is the primary approach in protecting livestock against the disease through a strong cellular immune response. Attenuated vaccine strains also induce serum anti-Brucella antibodies, mostly against Brucella O-polysaccharide, but their role in protection against the disease remains unclear. In this study, we show that Brucella OPS serum antibodies after vaccination or natural infection could kill Brucella in vitro as shown by the serum bactericidal activity (SBA) assay. We used serum samples of Rev. 1 vaccinated sheep that were negative or positive for Brucella OPS antibodies by either one of complement fixation test (CFT), microplate agglutination test (MAT) and ELISA, or sera of naturally infected sheep positive by CFT. We found a significant increase in the killing ability of sera 30 days after intraocular vaccination with Rev. 1 as compared with pre-vaccination. SBA was significantly higher in sera containing Brucella OPS IgG antibodies in comparison with sera lacking such antibodies (p < 0.001 against 16M & Rev. 1 strains). All 10 sera of convalescent sheep demonstrated significant killing ability against the 16M B. melitensis field strain. Specific OPS antibodies participate in the in vitro complement mediated Brucella killing suggesting a potential role in protection against the disease through this mechanism and relevance of developing OPS-based Brucella vaccines.

01. Serum Bactericidal Activity Against Circulating and Reference Strains of Meningococcal Serogroup B in the United States: A Review of Meningococcal Serogroup B (MenB) Vaccines in Adolescents and Young Adults

BackgroundUS adolescents and young adults are at particular risk of invasive meningococcal disease (IMD). In 2018, menincococcal serogroup B was responsible for 36% of IMD cases in the US overall and for 66% of cases in adolescents and young adults. This age group is at high risk of IMD during outbreaks, which result in significant response-related costs. MenB vaccine efficacy against IMD relies on its ability to provide broad protection against diverse disease-causing strains. MenB-FHbp (Trumenba) and MenB-4C (Bexsero) are MenB vaccines licensed in the US as 2-dose series with an interval of 6 mo or 1 mo, respectively, recommended in healthy adolescents and young adults. We review available data on vaccine coverage of serogroup B strains.MethodsA literature review identified relevant information from peer-reviewed publications, congress presentations, and ClinicalTrials.gov. Previously presented but unpublished data from phase 2/3 studies were included.ResultsAfter 2 MenB-FHbp doses, percentages of adolescents and young adults achieving serum bactericidal activity assay using human complement (hSBA) titers ≥1:8 were 79%–99% for 4 heterologous representative test strains and 71%–97% for 10 additional strains, confirming cross-protection against a diverse strain panel (Figure 1; unpublished data). These 14 heterologous strains collectively represent ~80% of disease-causing strains in the US and Europe. In a published study with limited sample size, 44%–78% of subjects had hSBA titers ≥1:8 against strains from 4 US college outbreaks after 2 MenB-FHbp doses. After 2 MenB-4C doses, percentages of 10–25-year-olds achieving hSBA titers ≥1:5 against 3 reference strains homologous to the vaccine antigen were 82%–93% (published data); 15%–100% of adolescents achieved hSBA titers ≥1:4 against a panel of 14 strains (unpublished data). Of college students who received 2 MenB-4C doses, 53%–93% achieved hSBA titers ≥1:4 against 5 US outbreak strains (4/5 strains had antigenic similarity to MenB-4C; published data). ConclusionMenB-FHbp and MenB-4C protect against various serogroup B strains. As for the breadth of coverage provided by these vaccines, available data show that MenB-FHbp elicits robust immune responses to a wide variety of disease-causing strains prevalent in the US (Figure 2). Disclosures Tamera Coyne-Beasley, MD, MPH, Pfizer Inc and GlaxoSmithKline (Advisor or Review Panel member) Joseph Bocchini, MD, Pfizer Inc and Dynavax (Advisor or Review Panel member) Alejandro Cane, M.D., Pfizer Inc (Employee, Shareholder) Cindy Burman, PharmD, Pfizer Inc (Employee, Shareholder) Maria J. Tort, PhD, Pfizer Inc (Employee, Shareholder) Jessica Presa, MD, Pfizer Inc (Employee, Shareholder)

6. Pentavalent Meningococcal (MenABCWY) Vaccine is Safe and Well Tolerated With Immunogenicity Noninferior to Coadministered MenB-FHbp and MenACWY-CRM in a Phase 2 Study of Healthy Adolescents and Young Adults

BackgroundMeningococcal serogroups A, B, C, W and Y cause nearly all meningococcal disease globally. Vaccination is complicated by different dosing recommendations for serogroup B (MenB) and quadrivalent (MenACWY) vaccines, which could be solved with a single pentavalent vaccine. This study in adolescents and young adults evaluated a new pentavalent MenABCWY vaccine that combines 2 licensed vaccines, MenB-FHbp (Trumenba®; bivalent rLP2086) and MenACWY-TT (Nimenrix®), into a single vaccine.MethodsIn this ongoing, randomized, controlled, observer-blinded, multicenter study (NCT03135834), MenB vaccine-naive and MenACWY-naive or -experienced healthy 10–25-year-olds were randomized 1:2 to MenABCWY (Month 0,6) or MenB-FHbp (Month 0,6) and MenACWY-CRM (Month 0). Immune responses were measured by serum bactericidal activity assays with human complement (hSBA) against serogroup A, C, W and Y strains and 4 diverse, vaccine-heterologous MenB strains. Endpoints included percentages of subjects achieving ≥ 4-fold rises in titers from baseline. Noninferiority of immune responses was assessed at the 10% margin (95% CI lower limit > −10%). Safety was assessed.ResultsFollowing dose 2, high percentages of MenABCWY (n=543) and MenB-FHbp (n=1057) recipients achieved ≥ 4-fold rises against each of the 4 MenB strains (75.8−94.7% vs 67.4−95.0%) and titers reaching at least the lower limit of quantification against all 4 strains combined (79.9% vs 74.3%; Figure 1A). MenABCWY was noninferior to MenB-FHbp for all 5 endpoints. MenABCWY was also noninferior to a single MenACWY-CRM dose with 75.5−96.9% and 93.0−97.4% of MenABCWY recipients after dose 1 or 2, respectively, achieving ≥ 4-fold rises against serogroup A, C, W and Y depending on prior MenACWY experience (Figure 1B). Local reactions and systemic events after MenABCWY or MenB-FHbp were similarly frequent, mostly mild/moderate in severity (Figure 2), and unaffected by MenACWY experience.Figure 1. Immune Responses as Measured in hSBA to (A) MenB Test Strains at 1 Month After Dose 2 and (B) MenA, MenC, MenW, and MenY Test Strains at 1 Month After Doses 1 and 2 Figure 2. (A) Local Reactions and (B) Systemic Events Reported Within 7 Days After Any Dose ConclusionMenABCWY 4-fold immune responses from baseline were robust and noninferior to MenB-FHbp and MenACWY-CRM administered separately. Vaccination was safe and well tolerated. The favorable benefit-risk profile supports further MenABCWY development as a simplified alternative to current meningococcal vaccination practices. Funded by Pfizer.Disclosures James Peterson, MD, Pfizer (Scientific Research Study Investigator) Daniel Drazan, MD, Pfizer (Scientific Research Study Investigator) Hanna Czajka, MD, PhD, Pfizer (Scientific Research Study Investigator) Jason Maguire, MD, Pfizer (Employee, Shareholder) Jean-Louis Pregaldien, MS, Pfizer (Employee, Shareholder) Ilkka Seppa, MD, Pfizer (Scientific Research Study Investigator) Roger Maansson, MS, Pfizer (Employee, Shareholder) Robert O’Neill, PhD, Pfizer (Employee, Shareholder) Annaliesa S. Anderson, PhD, Pfizer (Employee, Shareholder) Paul Balmer, PhD, Pfizer Inc (Employee, Shareholder) Johannes Beeslaar, MD, Pfizer (Employee, Shareholder) John L. Perez, MD, MA, Pfizer Inc (Employee, Shareholder)

Bactericidal antibodies against hypervirulent Neisseria meningitidis C field strains following MenC-CRM or MenACWY-CRM priming and MenACWY-CRM booster in children

ABSTRACT An increase in invasive meningococcal disease (IMD) incidence was observed in Tuscany in 2015/2016, mainly due to hypervirulent clonal complex (cc) 11 strains. In a post-hoc analysis, we assessed bactericidal activity of antibodies in sera from children primed with MenACWY-CRM or MenC-CRM conjugate vaccines and receiving a MenACWY-CRM booster dose against 5 meningococcal C (MenC) strains isolated from IMD cases. Sera collected from 90 infants/toddlers who participated in a phase III, open-label study (NCT00667602) and its extension (NCT01345721) were tested by serum bactericidal activity assay with human complement (hSBA). Children were primed with either MenACWY-CRM at 6–8 and 12 months of age (group 2_MenACWY; N = 30), MenACWY-CRM (group 1_MenACWY; N = 30), or MenC-CRM at 12 months of age (group 1_MenC; N = 30); all received MenACWY-CRM booster dose at 22–45 months of age. Four tested strains (FI001–FI004) were C:P1.5–1,10-8:F3-6:ST-11 (cc11) and 1 (FI005) was C:P1.7–4,14-6:F3-9:ST-1031 (cc334). Overall, immune responses tended to be higher against Fl002–FI004 than Fl001 and Fl005. Geometric mean titers were high in group 2_MenACWY (range: 94.8 [FI005]–588.1 [FI004]) and very high post-boosting with MenACWY-CRM in all groups (176.9 [FI005]–3911.0 [FI004]). Seroresponse rates tended to be higher in group 1_MenC (33.3% [FI005]–93.3% [FI004]) than in group 1_MenACWY (16.7% [FI005]–73.3% [FI004]). Irrespective of strains tested or the identity/number of priming doses, ≥96.7% of children had hSBA titers ≥1:8 post-MenACWY-CRM booster dose. MenACWY-CRM and MenC-CRM elicited bactericidal antibodies and immunological memory against hypervirulent cc11 and cc334 MenC strains responsible for IMD outbreaks.

Immunogenicity study of OmpU subunit vaccine against Vibrio mimicus in yellow catfish, Pelteobagrus fulvidraco

The outer membrane protein U (OmpU) is a conserved outer membrane protein in a variety of pathogenic Vibrio species and has been considered as a vital protective antigen for vaccine development. Vibrio mimicus (V. mimicus) is the pathogen causing ascites disease in aquatic animals. In this study, the prokaryotically expressed and purified His-tagged OmpU of V. mimicus (His-OmpU) was used as a subunit vaccine. The formalin inactivated V. mimicus, purified His tag (His-tag), and PBS were used as controls. The vaccinated yellow catfish were challenged with V. mimicus at 28 days post-vaccination, and the results showed that the His-OmpU and inactivated V. mimicus groups exhibited much higher survival rates than the His-tag and PBS groups. To fully understand the underlying mechanism, we detected the expression levels of several immune-related genes in the spleen of fish at 28 days post-vaccination and 24 h post-challenge. The results showed that most of the detected immune-related genes were significantly upregulated in His-OmpU and inactivated V. mimicus groups. In addition, we performed the serum bactericidal activity assay, and the results showed that the serum from His-OmpU and inactivated V. mimicus groups exhibited much stronger bactericidal activity against V. mimicus than those of His-tag and PBS groups. Finally, the serum agglutination antibody was detected, and the antibody could be detected in His-OmpU and inactivated V. mimicus groups with the antibody titers increasing along with the time post-vaccination, but not in His-tag or PBS group. Our data reveal that the recombinant OmpU elicits potent protective immune response and is an effective vaccine candidate against V. mimicus in yellow catfish.

Immune status of Oreochromis niloticus subjected to long-term lead nitrate exposure and a Arthrospira platensis treatment trial

In this study, the impacts of lead toxicity on Oreochromisniloticus were investigated. Additionally, the potential ameliorative effects of the Spirulina algae Arthrospira platensis were evaluated. The median lethal concentration (LC50) of PbNO3 was determined to be 143.3 mg/l for O. niloticus weighing 42 ± 2.5 g. O. niloticus were exposed to 10 % of the estimated PbNO3 LC50 for 12 weeks. The cumulative mortality rate (CMR) increased with exposure time. The results of assays for red blood cells (RBCs), haemoglobin (Hb), packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), and mean corpuscular haemoglobin concentration (MCHC) indicated that the exposed O. niloticus suffered from anaemia. The levels of liver enzymes, namely, aspartate transaminase (AST) and alanine transaminase (ALT), as well as metallothionein)MT(revealed deterioration of hepatic tissue. The activity of the antioxidant enzymes glutathione peroxidase (GPx) as well as catalase (CAT) was stimulated in the hepatic tissue of O. niloticus exposed to PbNO3 and in those treated with A. platensis. Based on the results of serum bactericidal activity (SBA) and oxidative burst activity (OBA) assays as well as challenge tests with Aeromonas hydrophila, it was clear that supplementation with 5 or 10 g/kg A. platensis significantly enhanced the fish immune status and decreased the mortality rate (MR). However, these effects were reduced by PbNO3 exposure with no differences in MR percentage. Therefore, it was clear that O. niloticus reared in lead nitrate-polluted water were immunosuppressed, while diet supplementation with A. platensis could ameliorate such impacts.

Pathogenicity of an Invasive Bacterium, Aeromonas salmonicida in Indian Major Carp, Labeo rohita – Evaluation of Immune Response Using Effective Molecular Markers

Effects of Aeromonas salmonicida on the non-specific immune response, oxidative stress and subsequent cell death in liver and spleen of Labeo rohita exposed to asymptomatic dose (2 × 107 CFU/mL) of pathogen were studied. Evaluation of in vivo non-specific immunity via Nitro blue tetrazolium (NBT) reduction assay, myeloperoxidase (MPO) assay and serum bactericidal activity assay exhibited significant alterations among infected fishes, indicating temporary surge of innate immune responses. Considerable cell death and damage was observed in the histopathological analysis of liver and spleen in the infected fishes. Flow cytometric analysis by FITC-Annexin V/PI conclusively indicated triggering of apoptotic cell death in both organs. Relative expression of pro-inflammatory cytokine (TNFα) gene, antimicrobial protein producing genes (Lysozyme C and Lysozyme G) and apoptosis effector molecule (Caspase 3) gene were increased significantly both in liver and spleen of A. salmonicida treated fishes. In conclusion, infection with A. salmonicida at asymptomatic level resulted in severe oxidative stress that, caused apoptotic cell death. Since facultative pathogen eradication is not possible in open water or flowthrough aquaculture systems, this remains a crucial area of scientific research and the results obtained in the present study might provide better understanding of prophylactic, diagnostic and therapeutic measures during aquaculture.

2722 B. Efficacy and Safety of a Booster Dose of the MenACWY-TT Vaccine Administered 10 Years After Primary Vaccination with MenACWY-TT or MenACWY-PS

BackgroundThe quadrivalent meningococcal ACWY polysaccharide tetanus toxoid conjugate vaccine (MenACWY-TT; Nimenrix) is licensed in various countries to prevent disease caused by meningococcal serogroups A, C, W, and Y. In a previous study (NCT00464815), subjects aged 11‒17 years received a primary dose of MenACWY-TT or a quadrivalent polysaccharide vaccine (MenACWY-PS). Here, we report the long-term antibody persistence of the primary dose and the immunogenicity and safety of a booster dose given 10 years after primary vaccination of subjects.MethodsParticipants were enrolled from the Philippines and received a booster dose of MenACWY-TT at 10 years postvaccination. Antibody persistence 10 years postprimary vaccination and immunogenicity 1 month after the booster dose were evaluated by serum bactericidal activity assays using rabbit complement (rSBA) to assess the percentages of subjects with titers ≥ 1:8 and ≥ 1:128 and geometric mean titers (GMTs) for each serogroup. Safety was assessed for the booster dose.ResultsOf 229 subjects enrolled in this extension study, 169 and 58 subjects in the MenACWY-TT and MenACWY-PS groups, respectively, completed the booster phase. The percentages of primary MenACWY-TT recipients with prebooster rSBA titers ≥ 1:8 and ≥ 1:128 at year 10 ranged from 71.6%‒90.7% and 64.8%‒85.2% for all serogroups, respectively, compared with 43.1%‒82.4% and 25.5%‒76.5% of primary MenACWY-PS recipients; rSBA GMTs for all serogroups were higher in the MenACWY-TT group than in the MenACWY-PS group at year 10. For the MenACWY-TT and MenACWY-PS groups, respectively, the MenACWY-TT booster dose elicited rSBA titers ≥ 1:8 in 100% and ≥ 98.0% of subjects (figure); 100% and ≥ 96.1% of all subjects had titers ≥ 1:128. For all serogroups, rSBA GMTs at 1 month after the booster dose were higher than before the booster dose. No new safety signals were observed during the booster phase.ConclusionFunctional antibody responses elicited by MenACWY-TT persisted 10 years after primary vaccination; the booster dose was well tolerated and elicited robust immune responses.ClinicalTrials.govNCT03189745, EudraCT # 2013-001512-29. Funded by Pfizer. Disclosures All authors: No reported disclosures.

1478. Efficacy and Safety of a Booster Dose of the MenACWY-TT Vaccine Administered 10 Years After Primary Vaccination with MenACWY-TT or MenACWY-PS

BackgroundThe quadrivalent meningococcal ACWY polysaccharide tetanus toxoid conjugate vaccine (MenACWY-TT; Nimenrix) is licensed in various countries to prevent disease caused by meningococcal serogroups A, C, W, and Y. In a previous study (NCT00464815), subjects aged 11‒17 years received a primary dose of MenACWY-TT or a quadrivalent polysaccharide vaccine (MenACWY-PS). Here, we report the long-term antibody persistence of the primary dose and the immunogenicity and safety of a booster dose given 10 years after primary vaccination of subjects.MethodsParticipants were enrolled from the Philippines and received a booster dose of MenACWY-TT at 10 years postvaccination. Antibody persistence 10 years postprimary vaccination and immunogenicity 1 month after the booster dose were evaluated by serum bactericidal activity assays using rabbit complement (rSBA) to assess the percentages of subjects with titers ≥1:8 and ≥1:128 and geometric mean titers (GMTs) for each serogroup. Safety was assessed for the booster dose.ResultsOf 229 subjects enrolled in this extension study, 169 and 58 subjects in the MenACWY-TT and MenACWY-PS groups, respectively, completed the booster phase. The percentages of primary MenACWY-TT recipients with prebooster rSBA titers ≥ 1:8 and ≥ 1:128 at year 10 ranged from 71.6%‒90.7% and 64.8%‒85.2% for all serogroups, respectively, compared with 43.1%‒82.4% and 25.5%‒76.5% of primary MenACWY-PS recipients; rSBA GMTs for all serogroups were higher in the MenACWY-TT group than in the MenACWY-PS group at year 10. For the MenACWY-TT and MenACWY-PS groups, respectively, the MenACWY-TT booster dose elicited rSBA titers ≥1:8 in 100% and ≥98.0% of subjects (figure); 100% and ≥96.1% of all subjects had titers ≥1:128. For all serogroups, rSBA GMTs at 1 month after the booster dose were higher than before the booster dose. No new safety signals were observed during the booster phase.ConclusionFunctional antibody responses elicited by MenACWY-TT persisted 10 years after primary vaccination; the booster dose was well tolerated and elicited robust immune responses.ClinicalTrials.gov: NCT03189745, EudraCT # 2013-001512-29. Funded by Pfizer. Disclosures All authors: No reported disclosures.

Multi-component meningococcal serogroup B (MenB)-4C vaccine induces effective opsonophagocytic killing in children with a complement deficiency.

SummaryVaccination against meningococcal serogroup B is recommended for patients with a complement deficiency; however, although immunogenicity in this patient group has been shown, efficacy has not yet been established. In this study, we collected serum from children with a complement deficiency in the alternative pathway or in late terminal pathway before and after vaccination with multi‐component meningococcal serogroup B (MenB)‐4C. MenB‐4C is a multi‐component, protein‐based vaccine against MenB consisting of factor H‐binding protein, Neisserial heparin‐binding protein, Neisserial adhesion A and outer membrane vesicles containing Porin A. We assessed the vaccine immunogenicity and vaccine‐mediated protection by a whole cell enzyme‐linked immunosorbent assay with Neisseria meningitidis serogroup B strains H44/76, 5/99 and NZ98/254, which shows that vaccination induced antibody titers against meningococcus. We show that the classical serum bactericidal activity assay with exogenous serum indicates the presence of vaccine‐induced antibodies and capacity to activate complement‐mediated pathogen lysis. However, in children with a late terminal pathway deficiency, no complement‐mediated pathogen lysis was observed when autologous serum was applied in the serum bactericidal activity assay, demonstrating a lack of serum bactericidal activity in children with complement deficiencies. However, MenB‐4C vaccination still induced effective complement‐dependent opsonophagocytic killing against N. meningitidis serogroup B in reconstituted whole blood with autologous serum from children with an alternative pathway or late terminal pathway deficiency. These findings support the recommendation to vaccinate all complement‐deficient children against MenB.

MenACWY-CRM conjugate vaccine booster dose given 4–6 years after priming: Results from a phase IIIb, multicenter, open label study in adolescents and adults

BackgroundVaccination strategies against bacterial meningitis vary across countries. In the United States, a single dose of quadrivalent meningococcal conjugate vaccine (MenACWY) is recommended at 11–12 years of age, with a booster dose approximately 5 years later. We assessed immune responses to a booster dose of MenACWY-CRM vaccine after priming with MenACWY-CRM or MenACWY-D vaccines in adolescents and adults. MethodsIn this phase IIIb, multicenter, open-label study, healthy 15–55-year-olds, who received MenACWY-CRM (N = 301) or MenACWY-D (N = 300) 4–6 years earlier or were meningococcal vaccine-naïve (N = 100), received one MenACWY-CRM vaccine dose. Immunogenicity was evaluated pre-vaccination, 3 or 5 days post-vaccination (sampling subgroups), and 28 days post-vaccination by serum bactericidal activity assay using human complement (hSBA). After vaccination, participants were monitored for 7 days for reactogenicity, 29 days for unsolicited adverse events (AEs), and 181 days for serious AEs and medically-attended AEs. ResultsSufficiency of the immune response to a MenACWY-CRM booster dose was demonstrated; the lower limit of the 1-sided 97.5% confidence interval for percentages of participants with hSBA seroresponse at 28 days post-vaccination was >75% for each serogroup in those primed with either the MenACWY-CRM or MenACWY-D vaccine. Seroresponse was observed in ≥93.24% of primed participants and ≥35.87% of naïve participants 28 days post-vaccination. At 5 days post-booster, among primed participants, hSBA titers ≥1:8 were achieved in ≥47.14% of participants for MenA and in ≥85.52% of participants for MenC, MenW and MenY, and 3.25- to 8.59-fold increases in hSBA geometric mean titers against each vaccine serogroup were observed. No safety concerns were raised throughout the 6-month follow-up period. ConclusionsA booster dose of the MenACWY-CRM vaccine induced a robust and rapid anamnestic response in adolescents and adults, irrespectively of either MenACWY-CRM or MenACWY-D vaccine administered 4–6 years earlier, with an acceptable clinical safety profile.ClinicalTrials.gov registration: NCT02986854.An Audio Summary linked to this article that can be found on Figshare https://figshare.com/articles/MenACWY-CRM_conjugate_vaccine_booster_dose_given_4_6_years_after_priming_Results_from_a_phase_IIIb_multicenter_open_label_study_in_adolescents_and_adults_mp4/11823048

The Development of a Vaccine Against Meningococcus B Using Reverse Vaccinology.

The discovery of vaccine antigens through whole genome sequencing (WGS) contrasts with the classical hypothesis-driven laboratory-based analysis of microbes to identify components to elicit protective immunity. This radical change in scientific direction and action in vaccine research is captured in the term reverse vaccinology. The complete genome sequence of an isolate of Neisseria meningitidis serogroup B (MenB) was systematically analyzed to identify proteins predicted to be secreted or exported to the outer membrane. This identified hundreds of genes coding for potential surface-exposed antigens. These were amplified, cloned in expression vectors and used to immunize mice. Antisera against 350 recombinant antigens were obtained and analyzed in a panel of immunological assays from which 28 were selected as potentially protective based on the -antibody dependent, complement mediated- serum bactericidal activity assay. Testing of these candidate vaccine antigens, using a large globally representative strain collection of Neisseria species isolated from cases of disease and carriage, indicated that no single component would be sufficient to induce broad coverage and that a “universal” vaccine should contain multiple antigens. The final choice of antigens to be included was based on cross-protective ability, assayed by serum bactericidal activity and maximum coverage of the extensive antigenic variability of MenB strains. The resulting multivalent vaccine formulation selected consisted of three recombinant antigens (Neisserial Heparin Binding Antigen or NHBA, Factor H binding protein or fHbp and Neisseria Adhesin A or NadA). To improve immunogenicity and potential strain coverage, an outer membrane vesicle component obtained from the epidemic New Zealand strain (OMVNz) was added to the formulation to create a four component vaccine, called 4CMenB. A series of phase 2 and 3 clinical trials were conducted to evaluate safety and tolerability and to estimate the vaccine effectiveness of human immune responses at different ages and how these were affected by various factors including concomitant vaccine use and lot-to-lot consistency. 4CMenB was approved in Europe in 2013 and introduced in the National Immunization Program in the UK starting from September 2015 when the vaccine was offered to all newborns using a 2, 4, and 12 months schedule., The effectiveness against invasive MenB disease measured at 11 months after the study start and 5 months after the second vaccination was 83% and there have been no safety concerns.

One-year antibody persistence and safety of a 4-dose schedule of MenACWY-CRM in healthy infants from South Korea.

PurposeResults from a post-marketing study to generate evidence on 1-year antibody persistence and safety following vaccination of infants from South Korea with the quadrivalent meningococcal conjugate vaccine MenACWY-CRM.Materials and MethodsIn this phase IV, open-label, multi-center study (NCT02446691), 128 infants received MenACWY-CRM at ages 2, 4, 6, and 12 months. One-year antibody persistence following the full vaccination course was evaluated (primary objective) for the four meningococcal serogroups (Men) by serum bactericidal activity assay using human or rabbit complement (hSBA/rSBA). Immune responses at 1-month post-vaccination and safety were also assessed.ResultsThe percentage of children with hSBA titers ≥8 ranged between 94% (MenA) and 100% (MenY/W) 1-month post-vaccination, and from 39% (MenA) to 89% (MenY) 1-year post-vaccination. At least 99% and 92% of children had rSBA titers ≥8 and ≥128 against each meningococcal serogroup, 1-month post-vaccination. One-year post-vaccination, the percentage of children with rSBA titers ≥8 and ≥128 ranged from 54% (MenC) to 99% (MenA) and from 30% (MenC) to 98% (MenA). Geometric mean titers declined from 1-month to 1-year post-vaccination, when they varied between 6.8 (MenA) and 53.6 (MenW) by hSBA and between 17.2 (MenC) and 2,269.5 (MenA) by rSBA. At least one solicited and unsolicited adverse event was reported for 79% and 66% of children. Of 36 serious adverse events reported, none were vaccination-related.ConclusionAntibody persistence (hSBA/rSBA titers ≥8) was determined in 39%–99% of children 1 year after a 4-dose MenACWY-CRM series during infancy, with an acceptable clinical safety profile.

Immunogenicity and safety of the quadrivalent meningococcal ACWY-tetanus toxoid conjugate vaccine (MenACWY-TT) in splenectomized or hyposplenic children and adolescents: Results of a phase III, open, non-randomized study

BackgroundIndividuals with functional or anatomic asplenia are at high risk for meningococcal disease. We evaluated the immunogenicity and safety of 1 and 2 doses of the quadrivalent meningococcal serogroups A, C, W, Y tetanus toxoid-conjugate vaccine (MenACWY-TT) in this high-risk population. MethodsThis phase III, open-label, controlled, non-randomized study (NCT01641042) enrolled 1–17-year-olds with impaired splenic activity (high-risk group) and age-matched healthy controls (control group). We measured immune responses to MenACWY-TT by serum bactericidal activity assays using rabbit (rSBA) and human (hSBA) complement and in terms of antibodies against polysaccharides of the 4 vaccine serogroups. We evaluated vaccine response rates (VRRs) as 4-fold increases from pre-vaccination levels or titers ≥1:32 (rSBA)/≥1:8 (hSBA). We recorded solicited and unsolicited adverse events (AEs) during 4 and 31 days post-vaccination, and serious AEs (SAEs) and new onset of chronic illnesses (NOCIs) throughout the study. ResultsThe according-to-protocol cohort for immunogenicity included 40 participants per group. In both groups, the first MenACWY-TT dose induced rSBA VRRs of 92.5–100% and hSBA VRRs of 55.6–77.1% across vaccine serogroups. Following the second MenACWY-TT dose, all participants had high responses, with rSBA and hSBA VRRs of 73.0–100% across vaccine serogroups. rSBA and hSBA geometric mean titers for each serogroup increased in both groups (with different magnitudes, but ≥13.1-fold) compared with baseline levels. Polysaccharide antibody concentrations ≥2.0 μg/ml were detected in ≥84.4% of participants and were similar between groups. Incidences of solicited and unsolicited AEs were comparable between groups. We recorded SAEs in 4/43 participants in the high-risk group and 1/43 participants in the control group (none vaccine-related). No NOCIs were reported. ConclusionIn this descriptive study, MenACWY-TT induced similar functional and humoral immune responses and had a clinically acceptable safety profile in children and adolescents with impaired splenic activity and in healthy controls.

Recombinant NADP-dependent isocitrate dehydrogenase of Edwardsiella tarda induces both Th1 and Th2 type immune responses and evokes protective efficacy against edwardsiellosis

Edwardsiella tarda has become one of the most severe fish pathogens throughout the world. Thus, studies on the design and production of highly protective vaccines against this pathogen, as well as the mechanisms of vaccine-induced disease resistance are urgently needed. In this study, the NADP-dependent isocitrate dehydrogenase (IDH) of E. tarda was recombinantly expressed and flounder (Paralichthys olivaceus) anti-rIDH serum was prepared. Also, the outer membrane proteins (OMPs) of E. tarda were extracted and analyzed by western blotting. The results showed that flounder anti-rIDH serum could specifically recognize a 44 kDa protein of E. tarda OMPs, which was identified to be the native IDH of E. tarda by mass spectrometric. Consistently, immunogold electron microscopy showed that IDH could be detected on the membrane of E. tarda. Then, the vaccine potential of rIDH was tested in a flounder model, and the results showed that rIDH produced a relative percent survival (RPS) of 73.3%, which was significantly higher than that produced by formalin killed E. tarda cells. Immunological analysis showed that rIDH could induce the proliferation of rIDH-specific sIg+ lymphocytes, which resulted in the production of anti-E. tarda antibodies. Accordingly, serum bactericidal activity assay showed that the serum of rIDH vaccinated fish exhibited the highest bactericidal activity compared with other groups. qRT-PCR analysis showed that rIDH could enhance the expressions of IFN-γ, NKEF, IL-6, MHCIα, CD4-1 and CD8α. Moreover, the bacterial burden was also detected in vaccinated fish after challenge, which showed that the number of E. tarda cells in spleen of rIDH group was significantly lower than other groups. All these results suggested that rIDH is a promising vaccine candidate against E. tarda infection.

Outer membrane vesicles extracted from Neisseria meningitidis serogroup X for prevention of meningococcal disease in Africa

Meningococcal disease is caused mainly by serogroups A, B, C, Y, W of N. meningitidis. However, numerous cases of meningitis caused by serogroup X N. meningitidis (MenX) have recently been reported in several African countries. Currently, there are no licensed vaccines against this pathogen and most of the MenX cases have been caused by meningococci from clonal complex (c.c) 181. Detergent extracted meningococcal outer membrane vesicle (dOMV) vaccines have previously shown to be safe and effective against epidemics of serogroup B meningococcal disease in all age groups. The aim of this work is therefore to obtain, characterize and evaluate the vaccine potential of dOMVs derived from a MenX strain (OMVx). Three experimental lots of OMVx were prepared by deoxycholate extraction from the MenX strain BF 2/97. Size and morphology of the vesicles was determined by Dynamic Light Scattering and electron microscopy, whereas the antigenic composition was characterized by gel electrophoresis and immunoblotting. OMVx were thereafter adsorbed to aluminium hydroxide (OMVx/AL) and two doses of OMVx were administered s.c. to groups of Balb/c mice three weeks apart. The immunogenicity and functional antibody activities in sera were evaluated by ELISA (anti-OMVx specific IgG responses) and serum bactericidal activity (SBA) assay. The size range of OMVx was shown to be between 90 and 120nm, whereas some of the antigens detected were the outer membrane proteins PorA, OpcA and RmpM. The OMVx/AL elicited high anti-OMVx antibody responses with bactericidal activity and no bactericidal activity was observed in the control group of no immunised mice. The results demonstrate that OMVx are immunogenic and could form part of a future vaccine to prevent the majority of meningococcal disease in the African meningitis belt.

Development of a high-throughput method to evaluate serum bactericidal activity using bacterial ATP measurement as survival readout.

Serum Bactericidal Activity (SBA) assay is the method of choice to evaluate the complement-mediated functional activity of both infection- and vaccine-induced antibodies. To perform a typical SBA assay, serial dilutions of sera are incubated with target bacterial strains and complement. The conventional SBA assay is based on plating on agar the SBA reaction mix and counting the surviving bacterial colony forming units (CFU) at each serum dilution. Even with automated colony counting, it is labor-intensive, time-consuming and not amenable for large-scale studies. Here, we have developed a luminescence-based SBA (L-SBA) method able to detect surviving bacteria by measuring their ATP. At the end of the SBA reaction, a single commercially available reagent is added to each well of the SBA plate, and the resulting luminescence signal is measured in a microplate reader. The signal obtained is proportional to the ATP present, which is directly proportional to the number of viable bacteria. Bactericidal activity is subsequently calculated. We demonstrated the applicability of L-SBA with multiple bacterial serovars, from 5 species: Citrobacter freundii, Salmonella enterica serovars Typhimurium and Enteritidis, Shigella flexneri serovars 2a and 3a, Shigella sonnei and Neisseria meningitidis. Serum bactericidal titers obtained by the luminescence readout method strongly correlate with the data obtained by the conventional agar plate-based assay, and the new assay is highly reproducible. L-SBA considerably shortens assay time, facilitates data acquisition and analysis and reduces the operator dependency, avoiding the plating and counting of CFUs. Our results demonstrate that L-SBA is a useful high-throughput bactericidal assay.

Human Infant Memory B Cell and CD4+ T Cell Responses to HibMenCY-TT Glyco-Conjugate Vaccine.

Carrier-specific T cell and polysaccharide-specific B cell memory responses are not well characterised in infants following glyco-conjugate vaccination. We aimed to determine if the number of Meningococcal (Men) C- and Y- specific memory B cells and; number and quality of Tetanus Toxoid (TT) carrier-specific memory CD4+ T cells are associated with polysaccharide-specific IgG post HibMenCY-TT vaccination. Healthy infants received HibMenCY-TT vaccine at 2, 4 and 6 months with a booster at 12 months. Peripheral blood mononuclear cells were isolated and polysaccharide-specific memory B cells enumerated using ELISpot. TT-specific memory CD4+ T cells were detected and phenotyped based on CD154 expression and intracellular TNF-α, IL-2 and IFN-γ expression following stimulation. Functional polysaccharide-specific IgG titres were measured using the serum bactericidal activity (SBA) assay. Polysaccharide-specific Men C- but not Men Y- specific memory B cell frequencies pre-boost (12 months) were significantly associated with post-boost (13 months) SBA titres. Regression analysis showed no association between memory B cell frequencies post-priming (at 6 or 7 months) and SBA at 12 months or 13 months. TT-specific CD4+ T cells were detected at frequencies between 0.001 and 0.112 as a percentage of CD3+ T cells, but their numbers were not associated with SBA titres. There were significant negative associations between SBA titres at M13 and cytokine expression at M7 and M12. Conclusion: Induction of persistent polysaccharide-specific memory B cells prior to boosting is an important determinant of secondary IgG responses in infants. However, polysaccharide-specific functional IgG responses appear to be independent of the number and quality of circulating carrier-specific CD4+ T cells after priming.

Honey bee pollen improves growth, immunity and protection of Nile tilapia (Oreochromis niloticus) against infection with Aeromonas hydrophila

The mode of action of honey bee pollen (HBP) was investigated in Nile tilapia (Oreochromis niloticus) challenged with Aeromonas hydrophila. Thus, fish with an average weight of 29 ± 3 g were divided into four groups, and fed with HBP-free diet (control), and 1%, 2.5% and 4% (w/v) HBP incorporated into basal diet for 10, 20 and 30 days. Immunological, hematological, biochemical and growth parameters were measured, and sub-groups of fish were challenged with A. hydrophila via intraperitoneal injection. HBP significantly increased the growth performance parameters [body weight, length, average daily gain (ADG), specific growth rate (SGR), and feed efficiency ratio (FER)] and immunological (phagocytic activity, serum bactericidal activity and nitroblue tetrazolium assay (NBT)), hematological (hematocrit (Hct), leucocrit (Lct), the numbers of neutrophils, monocytes and lymphocytes) and biochemical parameters (serum total protein, albumin and globulin ratios). Furthermore, all treated fish exhibited significant protection against challenge with A. hydrophila, with the highest protection (93%) observed in the group fed with 2.5% (w/v) HBP for 20 and 30 days.

Sequential Acquisition of T Cells and Antibodies to Nontyphoidal Salmonella in Malawian Children

Background. Salmonella Typhimurium (STm) remain a prominent cause of bacteremia in sub-Saharan Africa. Complement-fixing antibodies to STm develop by 2 years of age. We hypothesized that STm-specific CD4+ T cells develop alongside this process.Methods. Eighty healthy Malawian children aged 0–60 months were recruited. STm-specific CD4+ T cells producing interferon γ, tumor necrosis factor α, and interleukin 2 were quantified using intracellular cytokine staining. Antibodies to STm were measured by serum bactericidal activity (SBA) assay, and anti-STm immunoglobulin G antibodies by enzyme-linked immunosorbent assay.Results. Between 2006 and 2011, STm bacteremias were detected in 449 children <5 years old. STm-specific CD4+ T cells were acquired in infancy, peaked at 14 months, and then declined. STm-specific SBA was detectable in newborns, declined in the first 8 months, and then increased to a peak at age 35 months. Acquisition of SBA correlated with acquisition of anti–STm–lipopolysaccharide (LPS) immunoglobulin G (r = 0.329 [95% confidence interval, .552–.062]; P = .01) but not anti–STm–outer membrane protein or anti–STm-flagellar protein (FliC).Conclusions. Acquisition of STm-specific CD4+ T cells in early childhood is consistent with early exposure to STm or cross-reactive protein antigens priming this T-cell development. STm-specific CD4+ T cells seem insufficient to protect against invasive nontyphoidal Salmonella disease, but sequential acquisition of SBA to STm LPS is associated with a decline in its incidence.