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Abstract Thousands of disease cases and hundreds of deaths occur globally each year due to invasive meningococcal disease. Neisseria meningitidis serogroup B (MenB) is the leading cause of such disease in developed countries. Two vaccines, 4CMenB and MenB‐fHbp, that protect against MenB are available and include one or two forms respectively of factor H binding protein (fHbp), a key protective antigen. Studies of circulating meningococci have identified over 1380 different fHbp amino acid sequences, which form three immunologically distinct clusters, termed variants 1, 2, and 3. Neither of the current vaccines contains a variant 2 antigen, which is less well characterized than fHbp variants 1 and 3. We characterized the interaction of fHbp variant 2 with humAb 1B1 using biochemical methods and live meningococcal assays. Further, we determined the crystal structure of the complex at 2.4 Å resolution, clearly revealing the epitope and providing the first detailed report of an antibody with distinct specificity for fHbp variant 2. Extensive mutagenesis and binding studies elucidated key hotspots in the interface. This combination of structural and functional studies provides a molecular explanation for the bactericidal potency and specificity of humAb 1B1 for fHbp variant 2. Our studies, focused on fHbp variant 2, expand the understanding of this previously under characterized group of the vast family of variants of fHbp, a virulence factor present on all meningococci. Moreover, the definition of a protective conformational epitope on fHbp variant 2 may support the design and development of novel variant 2‐containing MenB vaccines affording greater breadth of protection.

Thousands of disease cases and hundreds of deaths occur globally each year due to invasive meningococcal disease. Neisseria meningitidis serogroup B (MenB) is the leading cause of such disease in developed countries. Two vaccines, 4CMenB and MenB‐fHbp, that protect against MenB are available and include one or two forms respectively of factor H binding protein (fHbp), a key protective antigen. Studies of circulating meningococci have identified over 1380 different fHbp amino acid sequences, which form three immunologically distinct clusters, termed variants 1, 2, and 3. Neither of the current vaccines contains a variant 2 antigen, which is less well characterized than fHbp variants 1 and 3. We characterized the interaction of fHbp variant 2 with humAb 1B1 using biochemical methods and live meningococcal assays. Further, we determined the crystal structure of the complex at 2.4 Å resolution, clearly revealing the epitope and providing the first detailed report of an antibody with distinct specificity for fHbp variant 2. Extensive mutagenesis and binding studies elucidated key hotspots in the interface. This combination of structural and functional studies provides a molecular explanation for the bactericidal potency and specificity of humAb 1B1 for fHbp variant 2. Our studies, focused on fHbp variant 2, expand the understanding of this previously under characterized group of the vast family of variants of fHbp, a virulence factor present on all meningococci. Moreover, the definition of a protective conformational epitope on fHbp variant 2 may support the design and development of novel variant 2‐containing MenB vaccines affording greater breadth of protection. [ABSTRACT FROM AUTHOR]

The increase in antimicrobial-resistant bacterial strains has highlighted the need for a new vaccine strategy. The primary goal of a candidate vaccine is to prevent disease, by inducing a persistent immunologic memory, through the activation of pathogen-specific immune response. Antibody titer is the main parameter used to assess the immunogenicity of bacterial vaccine candidates and it is the most widely used as a correlate of protection. On the other hand, the antibody titer alone cannot provide complete information on all the activity mediated by antibodies which can only be assessed by functional assays, like the serum bactericidal assay and the opsonophagocytosis assay. However, due to the involvement of many biological factors, these assays are difficult to standardize. Some improvements have been achieved in recent years, but further optimizations are needed to minimize inter- and intra-laboratories variability and to allow the applicability of these functional assays for the vaccine immunogenicity assessment on a larger scale. [ABSTRACT FROM AUTHOR]

The increase in antimicrobial-resistant bacterial strains has highlighted the need for a new vaccine strategy. The primary goal of a candidate vaccine is to prevent disease, by inducing a persistent immunologic memory, through the activation of pathogen-specific immune response. Antibody titer is the main parameter used to assess the immunogenicity of bacterial vaccine candidates and it is the most widely used as a correlate of protection. On the other hand, the antibody titer alone cannot provide complete information on all the activity mediated by antibodies which can only be assessed by functional assays, like the serum bactericidal assay and the opsonophagocytosis assay. However, due to the involvement of many biological factors, these assays are difficult to standardize. Some improvements have been achieved in recent years, but further optimizations are needed to minimize inter- and intra-laboratories variability and to allow the applicability of these functional assays for the vaccine immunogenicity assessment on a larger scale.

ABSTRACT: Introduction: There is scarce evidence in literature of what should be the best antimicrobial treatment for bloodstream infections (BSIs) sustained by Stenotrophomonas maltophilia, a peculiar pathogen that intrinsically withstands to most of the available antibiotics. Results and conclusion: Here, we describe a challenging case of a persistent S. maltophilia BSI due to septic thrombosis successfully treated with the addition of the novel siderophore cephalosporin cefiderocol to an only partially effective levofloxacin regimen. Additionally, an intra-lock therapy with trimethoprim/sulfamethoxazole was selected as a strategy to prevent recurrence of infection since complete source control was not possible. The serum bactericidal assay was also used to corroborate the in vivo efficacy of the adopted combination therapy.

Extensive efforts have been made toward improving effective strategies for pneumococcal vaccination, focusing on evaluating the potential of multivalent protein-based vaccines and overcoming the limitations of pneumococcal polysaccharide-based vaccines. In this study, we investigated the protective potential of mice co-immunization with the pneumococcal PhtD and novel rPspA proteins against pneumococcal sepsis infection. The formulations of each antigen alone or in combination were administered intraperitoneally with alum adjuvant into BALB/c mice three times at 14-day intervals. The production of antigen-specific IgG, IgG1 and IgG2a subclasses, and IL-4 and IFN-γ cytokines, were analyzed. Two in vitro complement- and opsonophagocytic-mediated killing activities of raised antibodies on day 42 were also assessed. Finally, the protection against an intraperitoneal challenge with 106 CFU/mouse of multidrug resistance of Streptococcus pneumoniae ATCC49619 was investigated. Our findings showed a significant increase in the anti-PhtD and anti-rPspA sera IgG levels in the immunized group with the PhtD+rPspA formulation compared to each alone. Moreover, the results demonstrated a synergistic effect with a 6.7-and 1.3- fold increase in anti-PhtD and anti-rPspA IgG1, as well as a 5.59- and 1.08- fold increase in anti-PhtD and anti-rPspA IgG2a, respectively. Coadministration of rPspA+PhtD elicited a mixture of Th-2 and Th-1 immune responses, more towards Th-2. In addition, the highest complement-mediated killing activity was observed in the sera of the immunized group with PhtD+rPspA at 1/16 dilution, and the opsonophagocytic activity was increased from 74% to 86.3%. Finally, the survival rates showed that mice receiving the rPspA+PhtD formulation survived significantly longer (100%) than those receiving protein alone or PBS and exhibited the strongest clearance with a 2 log10 decrease in bacterial load in the blood 24h after challenge compared to the control group. In conclusion, the rPspA+PhtD formulation can be considered a promising bivalent serotype-independent vaccine candidate for protection against invasive pneumococcal infection in the future. [ABSTRACT FROM AUTHOR]

Extensive efforts have been made toward improving effective strategies for pneumococcal vaccination, focusing on evaluating the potential of multivalent protein-based vaccines and overcoming the limitations of pneumococcal polysaccharide-based vaccines. In this study, we investigated the protective potential of mice co-immunization with the pneumococcal PhtD and novel rPspA proteins against pneumococcal sepsis infection. The formulations of each antigen alone or in combination were administered intraperitoneally with alum adjuvant into BALB/c mice three times at 14-day intervals. The production of antigen-specific IgG, IgG1 and IgG2a subclasses, and IL-4 and IFN-γ cytokines, were analyzed. Two in vitro complement- and opsonophagocytic-mediated killing activities of raised antibodies on day 42 were also assessed. Finally, the protection against an intraperitoneal challenge with 106 CFU/mouse of multi-drug resistance of Streptococcus pneumoniae ATCC49619 was investigated. Our findings showed a significant increase in the anti-PhtD and anti-rPspA sera IgG levels in the immunized group with the PhtD+rPspA formulation compared to each alone. Moreover, the results demonstrated a synergistic effect with a 6.7- and 1.3- fold increase in anti-PhtD and anti-rPspA IgG1, as well as a 5.59- and 1.08- fold increase in anti-PhtD and anti-rPspA IgG2a, respectively. Co-administration of rPspA+PhtD elicited a mixture of Th-2 and Th-1 immune responses, more towards Th-2. In addition, the highest complement-mediated killing activity was observed in the sera of the immunized group with PhtD+rPspA at 1/16 dilution, and the opsonophagocytic activity was increased from 74% to 86.3%. Finally, the survival rates showed that mice receiving the rPspA+PhtD formulation survived significantly longer (100%) than those receiving protein alone or PBS and exhibited the strongest clearance with a 2 log10 decrease in bacterial load in the blood 24h after challenge compared to the control group. In conclusion, the rPspA+PhtD formulation can be considered a promising bivalent serotype-independent vaccine candidate for protection against invasive pneumococcal infection in the future.

Objectives: Serum salmonellacidal (bactericidal) antibody could be used to detect functional capacity of antibody in patients with enteric fever and after typhoid vaccination. Methods: Salmonellacidal antibody response was measured by colorimetric serum salmonellacidal assay from 70 acute and 11 convalescence sera of patients infected with Salmonella Typhi and Paratyphi A and also from 15 control and 6 Vi capsular polysaccharide vaccinated volunteer's sera. Results: Sera from patients with typhoid and paratyphoid A showed significant (p < 0.05) levels of salmonellacidal antibody titer (549.9 ± 108.5 and 528.7 ± 187.3) compared with control (0.133 ± 0.1). Moreover, this titer increased significantly (p

Serum salmonellacidal (bactericidal) antibody could be used to detect functional capacity of antibody in patients with enteric fever and after typhoid vaccination. Salmonellacidal antibody response was measured by colorimetric serum salmonellacidal assay from 70 acute and 11 convalescence sera of patients infected with Salmonella Typhi and Paratyphi A and also from 15 control and 6 Vi capsular polysaccharide vaccinated volunteer's sera. Sera from patients with typhoid and paratyphoid A showed significant (p < 0.05) levels of salmonellacidal antibody titer (549.9 ± 108.5 and 528.7 ± 187.3) compared with control (0.133 ± 0.1). Moreover, this titer increased significantly (p <0.05) in sera collected between 7 and 10 days and between 11 and 25 days of fever (titer 535.7 ± 119.2 and 794.6 ± 235.6) compared with sera collected from patients with fever for less than 7 days (136.4 ± 52.7). The mean titer significantly (p < 0.05) decreased to 5.5 ± 2.1 after 6–8 weeks onset of illness. Although, very low salmonellacidal titers (2.5 ± 1.5 and 2.3 ± 1.5) were detected after Vi CPS vaccine among the human volunteers, but mean titer was raised 15-fold from pre- to postvaccinated sera (0.166–2.5). The serum salmonellacidal antibody by colorimetric salmonellacidal assay could be used to detect acute typhoidal cases and also to monitor immune response of typhoid vaccine. [ABSTRACT FROM AUTHOR]

Meningococcal meningitis is a rare but serious condition affecting mainly children and young adults. Outer membrane vesicles (OMV) from Neisseria meningitidis have been used successfully as vaccines against the disease, although they only provide protection against a limited number of the many existing variants. There have been many attempts to identify suitable protein antigens for use in defined vaccines that provide broad protection against the disease, such as that leading to the development of the four component 4CMenB vaccine. We previously reported the use of a protein antigen microarray to screen for IgG antibodies in sera derived from human recipients of an OMV-based vaccine, as part of a Phase I clinical trial. Here, we show that computational methods can be used to cluster antigens that elicit similar responses in the same individuals. Fitting of IgG antibody binding data to 4,005 linear regressions identified pairs of antigens that exhibited significant correlations. Some were from the same antigens in different quaternary states, whilst others might be correlated for functional or immunological reasons. We also conducted statistical analyses to examine correlations between individual serum bactericidal antibody (SBA) titres and IgG reactivity against specific antigens. Both Kendall's tau and Spearman's rank correlation coefficient statistics identified specific antigens that correlated with log(SBA) titre in five different isolates. The principal antigens identified were PorA and PorB, RmpM, OpcA, and the type IV pilus assembly secretin, PilQ. Other minor antigens identified included a lipoprotein, two proteins from the BAM complex and the efflux channel MtrE. Our results suggest that consideration of the entire antigen composition, and allowance for potential interaction between antigens, could be valuable in designing future meningococcal vaccines. Such an approach has the advantages that it uses data derived from human, rather than animal, immunization and that it avoids the need to screen individual antigens. [ABSTRACT FROM AUTHOR]

The four-component meningococcal serogroup B vaccine (4CMenB) contains antigens present in the majority of meningococci causing invasive meningococcal disease (IMD) and may potentially offer protection against strains belonging to non-B serogroups. This study aimed to evaluate the ability of 4CMenB-induced antibodies to kill, in a human serum bactericidal assay (hSBA), non-B meningococci belonging to the main genotypes responsible for IMD in Italy. Meningococci, collected between 2015 and 2017, was characterized for PorA, FetA and sequence type, and for clonal complex. Twenty non-B isolates, representative of the most frequent genotypes, were molecularly characterized for 4CMenB antigens and tested in hSBA with sera from 4CMenB-vaccinated infants and adolescents. Among twenty isolates, eleven were serogroup C, five were Y, two W and two X. All isolates contained genes encoding for fHbp and NHBA antigens and four harbored the NadA full-length encoding gene. Positive hSBA titers were obtained against all serogroup W, X and Y isolates and against five serogroup C isolates. These data show that the 4CMenB vaccine can induce bactericidal antibodies against genetically representative meningococcal W, Y and X strains from Italy. For serogroup C, different susceptibilities to killing were observed for strains with similar antigenic repertoires.

Serum bactericidal assay (SBA) is the method to investigate in vitro complement-mediated bactericidal activity of sera raised upon vaccination. The assay is based on incubating the target bacteria and exogenous complement with sera at different dilutions and the result of the assay is represented by the sera dilution being able to kill 50% of bacteria present in the inoculum. The traditional readout of the assay is based on measurement of colony-forming units (CFU) obtained after plating different reaction mixes on agar. This readout is at low throughput and time consuming, even when automated counting is used. We previously described a novel assay with a luminescence readout (L-SBA) based on measurement of ATP released by live bacteria, which allowed to substantially increase the throughput as well as to reduce the time necessary to perform the assay when compared to traditional methods. Here we present a further improvement of the assay by moving from a 96-well to a 384-well format, which allowed us to further increase the throughput and substantially reduce costs while maintaining the high performance of the previously described L-SBA method. The method has been successfully applied to a variety of different pathogens. [ABSTRACT FROM AUTHOR]

Introduction: Serum bactericidal assay is the gold standard index of protection against Vibrio cholerae. The outer membrane vesicles (OMVs) which are released during the bacterial growth have intrinsic immune stimulatory properties, based on their nature and composition. In this study, the induction of serum bactericidal activities in immunized BALB/c mice was determined using different vaccine regimens, using V. cholerae O1 (El Tor biotype) OMVs. Methods: A single clone of V. cholerae O1 (El Tor biotype) isolated during the 2005 outbreak in Iran, was used. A detergent-centrifugation procedure was used for OMVs production. Various vaccination regimens were inoculated into female mice via an oral route. The vaccine formulas included V. cholerae OMVs, killed whole cells of V. cholerae (WC), combination of WC-OMV and licensed cholera vaccine (Dukoral).The serum vibriocidal activity of mice sera was determined by measuring the complement-mediated lysis. Results: Electron microscopy of the purified OMVs from the isolated V. cholerae revealed the spherical-shaped vesicles of the size range 20-300 nm. In vitro reactivity of mice sera bactericidal capability against regimens of vaccination showed a significant immune response of antibody titers in comparison with negative control groups. Also, there was a significant increase in serum bactericidal titer of WC-OMV obtained from wild-type V. cholerae which had a satisfactory reactivity as Dukoral cholera vaccine. Conclusion: The results indicated that the combination of WC-OMV from the local strain is able to induce a high level of bactericidal antibody responses and it can be useful in optimization of the vaccine formula.

Vaccination is an effective strategy to combat invasive meningococcal disease (IMD). Vaccines against the major disease-causing meningococcal serogroups are available; however, development of vaccines against serogroup B faced particular challenges, including the inability to target traditional meningococcal antigens (i.e. polysaccharide capsule) and limited alternative antigens due to serogroup B strain diversity. Two different recombinant, protein-based, serogroup B (MenB) vaccines that may address these challenges are currently available. These vaccines have been extensively evaluated in pre-licensure safety and immunogenicity trials, and recently in real-world studies on effectiveness, safety, and impact on disease burden. This review provides healthcare professionals, particularly pediatricians, an overview of currently available MenB vaccines, including development strategies and evaluation of coverage. Overall, recombinant MenB vaccines are valuable tools for healthcare professionals to protect patients against IMD. Their development required innovative design approaches that overcame challenging hurdles and identified novel protein antigen targets; however, important distinctions in the approaches used in their development, evaluation, and administration exist and many unanswered questions remain. Healthcare providers frequently prescribing MenB vaccines are challenged to keep abreast of these differences to ensure patient protection against this serious disease. [ABSTRACT FROM AUTHOR]

Serum bactericidal assay (SBA) is the method to investigate in vitro complement-mediated bactericidal activity of sera raised upon vaccination. The assay is based on incubating the target bacteria and exogenous complement with sera at different dilutions and the result of the assay is represented by the sera dilution being able to kill 50% of bacteria present in the inoculum. The traditional readout of the assay is based on measurement of colony-forming units (CFU) obtained after plating different reaction mixes on agar. This readout is at low throughput and time consuming, even when automated counting is used. We previously described a novel assay with a luminescence readout (L-SBA) based on measurement of ATP released by live bacteria, which allowed to substantially increase the throughput as well as to reduce the time necessary to perform the assay when compared to traditional methods. Here we present a further improvement of the assay by moving from a 96-well to a 384-well format, which allowed us to further increase the throughput and substantially reduce costs while maintaining the high performance of the previously described L-SBA method. The method has been successfully applied to a variety of different pathogens.

Combination therapies are frequently used in the treatment of multidrug-resistant Klebsiella pneumoniae infection without consensus regarding which combination is the most effective. We compared bactericidal titres from sera collected from critically ill patients receiving meropenem plus tigecycline (n = 5), meropenem plus colistin (n = 5), or meropenem, colistin and tigecycline (n = 5) against K. pneumoniae isolates that included ESBL-producing (n = 7) and KPC-producing strains (n = 14) with varying sensitivity patterns to colistin and tigecycline. Meropenem concentrations (Cmin) were measured in all samples by LC-MS/MS, and indexed to respective pathogen MICs to explore differences in patterns of bactericidal activity for two versus three drug combination regimens. All combination regimens achieved higher SBTs against ESBL (median reciprocal titre 128, IQR 32–256) versus KPC (4, IQR 2–32) strains. Sera from patients treated with meropenem-colistin yielded higher median SBTs (256, IQR 64–512) than either meropenem-tigecycline (32, IQR 8–256; P < 0.001). The addition of tigecycline was associated with a lower probability of achieving a reciprocal SBT above 8 when meropenem concentrations were below the MIC (P = 0.04). Although the clinical significance is unknown, sera from patients receiving tigecycline-based combination regimens produce lower serum bactericidal titres against ESBL or KPC-producing K. pneumoniae. SBTs may represent a useful complimentary endpoint for comparing pharmacodynamics of combinations regimens for MDR Enterobacteriaceae. [ABSTRACT FROM AUTHOR]

Individuals suffering from severe chronic kidney disease (CKD) are immunocompromised and therefore highly susceptible to various infections including Haemophilus influenzae type a (Hia), an emerging pathogen in North American Indigenous populations. Immunocompromised Indigenous adults are considered a target for a new Hia vaccine under development. In an attempt to foresee their response to Hia immunization, we studied natural immunity against Hia and B-cell subpopulations in sixty patients with CKD residing in a geographic region with noticeable presence of Hia invasive disease. Serum bactericidal activity (SBA) against Hia, concentrations of IgG and IgM antibodies specific to Hia capsular polysaccharide, and B-cell subpopulations were studied in patients with CKD and 35 healthy controls of the same age. Of the patients with CKD, proportions and absolute numbers of B-cell subpopulations were determined for 28 patients. The patients had lower SBA titres compared to controls. Although no significant differences in anti-Hia IgG or IgM antibody concentrations between control and CKD groups were found, IgM antibody concentrations were higher in Indigenous than non-Indigenous patients. Patients with CKD had a higher proportion of B cells (CD19+), class switched memory B cells (CD19+CD27+IgM-) and a lower proportion of CD19+CD27-IgM- B cells compared to healthy controls. Non-Indigenous patients with CKD had significantly higher proportions of IgM memory B cells and CD19+CD27-IgM- B cells compared to Indigenous patients with no significant difference in absolute numbers. Because 72% of CKD patients had detectable SBA titres and 100% had detectable IgG and IgM antibodies it is possible that a portion of IgM memory B cells and class switched memory B cells are specific for Hia resulting from a natural exposure to the pathogen. The data suggest that a Hia-conjugate vaccine may be immunogenic in adult patients with CKD as it will potentially induce re-activation of immunological memory against Hia. [ABSTRACT FROM AUTHOR]

Background: Polysaccharide vaccines are effective in individuals from about the two years of age but, as they elicit T-cell independent immunity, they are not effective in younger children. In contrast, polysaccharide-protein conjugates are shown to be highly immunogenic in infants and induce T-cell dependent immunity. Methods: In this study, capsular polysaccharide of Neisseria meningitidis type A (NMA-CPS) was attached to recombinant protein of hepatitis B surface antigen (rHbsAg) covalently using amidation method. Immunization was done, choosing 2 groups of rabbits. Pure NMA-CPS and conjugated molecule were injected to groups 1 and 2, with a 15-day interval, intramuscularly. The bleeding was performed at days 0, 15, 30, 45 and titers of sera were measured via serum bactericidal assay. Findings: Polysaccharide bactericidal titer on days 15, 30 and 45 was almost identical and there was no increase in titer. But, in the first injection of the conjugate, the titer was much more (about twice of purified polysaccharide), and in the second injection, increased. Conclusion: Results display that conjugated molecules cause more immunity than pure capsular polysaccharide, and can stimulate cellular immunity.

Highlights • FHbp expression is not restricted to serogroup B strains of N. meningitidis. • Protection against non-MenB strains by antibodies elicited by the MenB-FHbp vaccine was studied. • hSBA response rate after dose 3 was ≥83% for MenC/W/Y/X and 28% for MenA strains. • MenB-FHbp may protect against meningococcal disease regardless of serogroup. Abstract MenB-FHbp (Trumenba®; bivalent rLP2086) is a meningococcal serogroup B vaccine containing 2 variants of the recombinant lipidated factor H binding protein (FHbp) antigen. The expression of FHbp, an outer membrane protein, is not restricted to serogroup B strains of Neisseria meningitidis (MenB). This study investigated whether antibodies elicited by MenB-FHbp vaccination also protect against non-MenB strains. Immunological responses were assessed in serum bactericidal assays using human complement (hSBAs) with non-MenB disease-causing test strains from Europe, Africa, and the United States. Importantly, FHbp variant distribution varies among meningococcal serogroups; therefore, strains that code for serogroup-specific prevalent variants (ie, representative of the 2 antigenically distinct FHbp subfamilies, designated subfamily A and subfamily B) and with moderate levels of FHbp surface expression were selected for testing by hSBA. After 2 or 3 doses of MenB-FHbp, 53% to 100% of individuals had bactericidal responses (hSBA titers ≥ 1:8) against meningococcal serogroup C, W, Y, and X strains, and 20% to 28% had bactericidal responses against serogroup A strains; in fact, these bactericidal responses elicited by MenB-FHbp antibodies against non-MenB strains, including strains associated with emerging disease, were greater than the serological correlate of protection for meningococcal disease (ie, hSBA titers ≥ 1:4). This is in comparison to a quadrivalent polysaccharide conjugate vaccine, MCV4 (Menactra®, targeting meningococcal serogroups A, C, W, and Y), which elicited bactericidal responses of 90% to 97% against the serogroup A, C, W, and Y strains and had no activity against serogroup X. Together, these results provide clinical evidence that MenB-FHbp may protect against meningococcal disease regardless of serogroup. [ABSTRACT FROM AUTHOR]

Increasing awareness of Salmonella Paratyphi A's contribution to enteric fever episodes throughout Asia has led to the development of new S. Paratyphi A vaccines. Assays are needed to measure functional antibodies elicited by the new vaccine candidates to assess their immunogenicity and potential protective capacities. Serum bactericidal assay (SBA) is the method of choice to measure functional antibody titers against various bacterial pathogens, but it is rarely been used for large dataset and clinical samples because it is time consuming and labor-intensive. Recently we developed a high-throughput luminescence-based SBA method, against different pathogens, including Salmonella Typhimurium and Enteritidis, Shigella flexneri serovars 2a and 3a, Shigella sonnei and Neisseria meningitidis . Here we further demonstrated the applicability of such method with invasive isolates of S. Paratyphi A to assess the complement-mediated antibody-dependent killing of both preclinical and clinical standard sera. As already found for other organisms, titers obtained by the luminescence-based SBA strongly correlated with those obtained by the conventional agar plate-based assay. The SBA assay described here is a useful tool for measuring functional antibodies elicited by Salmonella vaccines, with the potential of being applied to immunogenicity assessment in clinical trials. [ABSTRACT FROM AUTHOR]

The human serum is a vital component of the innate immunity of the host that acts as the first line of defence against invading pathogens. A key player in serum-mediated innate immune defence is a system of more than 35 proteins, collectively named as the complement system. After exposure of the pathogen, these proteins are activated in a cascade manner, ultimately forming a membrane attack complex (MAC) on the surface of the pathogen that directly lyses the bacterial cell. Formation of the MAC can be demonstrated in vitro by using serum bactericidal assay (SBA) that works in the absence of cellular components of blood after incubating the serum along with bacteria. Here, we describe the age-related differences in the bactericidal activity of human serum against Pseudomonas aeruginosa, an opportunistic human pathogen causing an array of hospital and community-acquired infections. We demonstrate that adult sera were highly effective in the in vitro killing of Pseudomonas aeruginosa as compared to children and the elderly (p < 0.0001). Sera from children were seriously compromised in the killing P. aeruginosa, whereas elderly sera showed a reduced level of killing. Data revealed a positive correlation between age and serum-killing with higher coefficient of determination values of 0.34, 0.27, and 0.58 and p values of < 0.0001, < 0.001, and < 0.0001, respectively, after 60, 90, and 120 minutes of incubation. Hence, our study highlights the age-related difference in the bactericidal activity of human sera. We conclude that sera of children are totally compromised, whereas elderly sera are only partially compromised, in the killing of P. aeruginosa. [ABSTRACT FROM AUTHOR]

Gonococcal microparticle vaccine in dissolving microneedle for transdermal delivery leading to generation of protective immunity and enhanced rate of clearance of infection in a preclinical animal model. Abbreviations: Gc-MP- gonorrhea vaccine microparticles, Gc-MP+ Alum MP+ AddaVax™ MP- adjuvanted vaccine microparticles. [Display omitted] There is an alarming rise in the number of gonorrhea cases worldwide. Neisseria gonorrhoeae , the bacteria that causes gonorrhea infection, has gradually developed antimicrobial resistance over the years. To date, there is no licensed vaccine for gonorrhea. This study investigates the in vivo immunogenicity of a whole-cell inactivated gonococci in a microparticle formulation (Gc-MP) along with adjuvant microparticles (Alhydrogel®- Alum MP and AddaVax™ MP) delivered transdermally using dissolving microneedles (MN). The proposed vaccine formulation (Gc-MP + Alum MP + AddaVax™ MP) was assessed for induction of humoral, cellular, and protective immune responses in vivo. Our results show the induction of significant gonococcal-specific serum IgG, IgG1, IgG2a, and vaginal mucosal IgA antibodies in mice immunized with Gc-MP + Alum MP + AddaVax™ MP and Gc-MP when compared to the control groups receiving blank MN or no treatment. The serum bactericidal assay revealed that the antibodies generated in mice after immunization with Gc-MP + Alum MP + AddaVax™ MP were bactericidal towards live Neisseria gonorrhoeae. Gc-MP + Alum MP + AddaVax™ MP and Gc-MP-immunized mice showed enhanced clearance rate of gonococcal bacterial infection post challenge. In contrast, the control groups did not begin to clear the infection until day 10. In addition, the mice which received Gc-MP + Alum MP + AddaVax™ MP showed enhanced expression of cellular immunity markers CD4 and CD8 on the surface of T cells in the spleen and lymph nodes. Taken together, the data shows that microneedle immunization with whole-cell inactivated gonococci MP in mice induced humoral, cellular, and protective immunity against gonococcal infection. [ABSTRACT FROM AUTHOR]

Background and aims Meningococcal C conjugate (MCC) vaccination programs provide direct and indirect protection against meningococcal disease. However, a decrease in the antibodies could affect herd immunity. We conducted a seroprevalence study to assess the immunity in subjects 8–12 years after different MCCV vaccination programs were launched and evaluated the impact of vaccination on seroprotection. Methods Seroepidemiological study conducted from October 2010 to April 2012 in the region of Valencia, Spain. Sample size was not proportional to the population but to the expected seroprotection by age group. Sera from subjects that were ≥ 3 years old were tested using a standardized complement-mediated serum bactericidal antibodies (SBA) assay. Age-stratified proportions of subjects with SBA titers ≥ 8 were considered seroprotected and evaluated. A multivariate logistic regression model was performed to evaluate the impact of vaccination on the seroprotection. Results Serum samples from 1880 subjects were collected. In total, 523 (27.8%) of the 1880 subjects and 446 (31.2%) of the 1430 subjects < 30 years (targeted to any vaccination campaign) showed protective SBA titers. The highest percentage of seroprotected subjects (67.8%, 95%CI 56.9–77.4) was observed in those that were vaccinated in a catch-up campaign at 10–13 years of age (20–21 years old at the time of blood sampling). Those scheduled for immunization in infancy at 2, 4 and 6 months of age (7–8 years at blood sample) represented the lowest (7.1%, 95% CI 3.3–13.1) number of seroprotected subjects. Having received one vaccine dose after 12 months of age was associated with increased seroprotection. The present study revealed a positive correlation between the increasing age at vaccination and longer duration of seroprotection. Conclusion Only one in three subjects who were vaccinated with MCC vaccine was seroprotected after 8–12 years. These findings emphasize that seroprevalence studies are essential to identify susceptible cohorts and to inform vaccine policy. [ABSTRACT FROM AUTHOR]

Hap, an auto-transporter protein, is an antigenically conserved adhesion protein which is present on both typeable and nontypeable Haemophilus influenzae . This protein has central role in bacterial attachment to respiratory tract epithelial cells. A 1000bp C-terminal fragment of Hap passenger domain (HapS) from nontypeable Haemophilus influenzae was cloned into a prokaryotic expression vector, pET-24a. BALB/c mice were immunized subcutaneously with purified rC-HapS. Serum IgG responses to purified rC-HapS, serum IgG subclasses were determined by ELISA and functional activity of antibodies was examined by Serum Bactericidal Assay. The output of rC-HapS was approximately 62% of the total bacterial proteins. Serum IgG responses were significantly increased in immunized group with rC-HapS mixed with Freund’s adjuvant in comparison with control groups. Analysis of the serum IgG subclasses showed that the IgG1 subclass was predominant after subcutaneous immunization in BALB/c mice (IgG2a/IgG1 < 1). The sera from rC-HapS immunized animals were strongly bactericidal against nontypeable Haemophilus influenzae . These results suggest that rC-HapS may be a potential vaccine candidate for nontypeable Haemophilus influenzae . [ABSTRACT FROM AUTHOR]

Typhoid fever, mainly caused by Salmonella enterica serovar Typhi ( S. Typhi), is a life-threatening disease, mostly in developing countries. Enzyme-linked immunosorbent assay (ELISA) is widely used to quantify antibodies against S. Typhi in serum but does not provide information about functional antibody titers. Although the serum bactericidal assay (SBA) using an agar plate is often used to measure functional antibody titers against various bacterial pathogens in clinical specimens, it has rarely been used for typhoid vaccines because it is time-consuming and labor-intensive. In the present study, we established an improved SBA against S. Typhi using a semi-automated colony-counting system with a square agar plate harboring 24 samples. The semi-automated SBA efficiently measured bactericidal titers of sera from individuals immunized with S. Typhi Vi polysaccharide vaccines. The assay specifically responded to S. Typhi Ty2 but not to other irrelevant enteric bacteria including Vibrio cholerae and Shigella flexneri . Baby rabbit complement was more appropriate source for the SBA against S. Typhi than complements from adult rabbit, guinea pig, and human. We also examined the correlation between SBA and ELISA for measuring antibody responses against S. Typhi using pre- and post-vaccination sera from 18 human volunteers. The SBA titer showed a good correlation with anti-Vi IgG quantity in the serum as determined by Spearman correlation coefficient of 0.737 ( P < 0.001). Taken together, the semi-automated SBA might be efficient, accurate, sensitive, and specific enough to measure functional antibody titers against S. Typhi in sera from human subjects immunized with typhoid vaccines. [ABSTRACT FROM AUTHOR]

Background: Non-typeable Haemophilus influenzae (NTHi) is a Gram negative microorganism residing in the human nasopharyngeal mucosa and occasionally causing infections of both middle ear and lower respiratory airways. A broadly protective vaccine against NTHi has been a long-unmet medical need, as the high genetic variability of this bacterium has posed great challenges. Results: In this study, we developed a robust serum bactericidal assay (SBA) to optimize the selection of protective antigens against NTHi. SBA takes advantage of the complement-mediated lysis of bacterial cells and is a key in vitro method for measuring the functional activity of antibodies. As a proof of concept, we assessed the bactericidal activity of antibodies directed against antigens known to elicit a protective response, including protein D used as carrier protein in the Synflorix pneumococcal polysaccharide conjugate vaccine. Prior to SBA screening, the accessibility of antigens to antibodies and the capacity of the latter to induce C3 complement deposition was verified by flow cytometry. Using baby rabbit serum as a source of complement, the proposed assay not only confirmed the bactericidal activity of the antibodies against the selected vaccine candidates, but also showed a significant reproducibility. Conclusions: Considering the rapidity and cost-effectiveness of this novel SBA protocol, we conclude that it is likely to become an important tool to prove the capability of antibodies directed against recombinant antigens to induce NTHi in vitro killing and to both select new protective vaccine candidates, and predict vaccine efficacy. [ABSTRACT FROM AUTHOR]

Abstract: Background: Neisseria meningitidis serogroup B (MnB) is a leading cause of bacterial meningitis and septicemia in adolescents and young adults. No currently licensed and available vaccine has been shown to provide broad protection against endemic MnB disease. A bivalent rLP2086 vaccine based on two factor H-binding proteins (fHBPs) has been developed to provide broad protection against MnB disease-causing strains. Methods: This study assessed the safety and immunogenicity of the final formulation of a bivalent rLP2086 vaccine in 60 healthy adults (18–40 years of age) receiving 120μg doses at 0, 1, and 6 months. Safety was assessed by collecting solicited reactogenicity data and participant-reporting of adverse events. Immunogenicity was evaluated by human serum bactericidal assay (hSBA) against 5 MnB strains expressing distinct fHBP variants and fHBP-specific immunoglobulin G titre. Results: After each immunisation, local reactions such as pain at the injection site and erythema were generally mild or moderate. The most common vaccine-related adverse event was upper respiratory tract infection, which was reported by two participants. Seroprotection (hSBA titres≥1:4) was achieved in 94.3% of participants against a MnB strain expressing the vaccine-homologous fHBP variant A05 and 70.0%–94.7% against MnB strains expressing the heterologous fHBP variants B02, A22, B44, and B24. Seroconversion rates (≥4-fold rise in hSBA titres) ranged from 70.0% to 94.7% across the five MnB test strains following the 3-dose vaccination regimen. Immunogenicity responses tended to increase upon subsequent vaccine doses. Conclusions: Bivalent rLP2086 is a promising vaccine candidate for broad protection against MnB disease-causing strains. [Copyright &y& Elsevier]

Abstract: Nontyphoidal isolates of Salmonella (NTS), particularly Salmonella Typhimurium, are a major cause of invasive bacteremia in Africa. Despite this, no vaccine against NTS is currently available for use in humans. If a NTS vaccine is to be developed in a timely manner, there is a need to develop assays to assess its in vivo efficacy. Assessment of potential efficacy of candidate vaccines in preclinical models is important for proof-of-concept and reduces attrition of vaccines in clinical trials. Serum bactericidal assays (SBA) are often used to assess the functional activity of vaccine-induced antibody responses targeted against Gram-negative bacteria with results given as the maximum dilution of serum that can effect bacterial killing. Previously we have found evidence for a protective role for antibody-induced complement-mediated killing of NTS in African children using an undiluted whole serum SBA. However, endogenous complement in diluted human sera is limiting and insufficient to effect bactericidal activity against S. Typhimurium beyond two two-fold dilutions. In the current study, we examined the requirements for SBA against NTS using baby rabbit serum (BRS) as an exogenous source of complement. We found that the amount of complement required for antibody-mediated bactericidal activity is much higher for the invasive African S. Typhimurium isolate D23580, compared with the laboratory S. Typhimurium LT2 and Salmonella Paratyphi A CVD1901. While 20% BRS was sufficient to kill LT2 and CVD1901, 75% BRS was needed to kill D23580. Our findings demonstrate that one concentration of exogenous complement is not suitable for SBA against all Salmonella isolates. To develop SBA to assess the in vivo efficacy of Salmonella vaccines, it is necessary to optimize the assay for the Salmonella isolates against which the vaccine is targeted. [Copyright &y& Elsevier]

A descriptive transversal study of N. meningitidis carriers involving 189 students of 12-19 years old of a Polytechnic School in Ciego de Avila province was carried out. This study aimed at knowing the prevalence of N. meningitidis carriers, the epidemiological markers as well as risk factors associated to carrier state and to basal immune response to VA-MENGOC-BC® in teenagers. Blood sample and pharyngeal swab tests were carried out to the students, in addition a survey was performed to find out aspects of research. Identification of N. meningitidis was carried out by API NH system (bioMérieux). Sero-subtypes and immunotypes were classified by whole cell ELISA with monoclonal antibodies, and basal immune response was detected by Serum Bactericidal Assay (SBA). Antimicrobial susceptibility of strains to Penicillin, Chloramphenicol, Rifampicin, Sodic Sulfadiazine, Ceftriaxone and Ciprofloxacine was determined. Prevalence of N. meningitidis was 17%. Non groupable strains were predominant (84.7%), followed by serogroups B (12.5%) and Z (3.1%), serogroup C was absent. Phenotype NA:NT:P1.NST:L3.7.9 (12.5%) prevailed, resistant strains to sulfadiazine (78.2%) and sensitive to penicillin (81.3%), though 18.7% showed intermediate sensitivity to this drug. All strains were sensitive to the remaining antimicrobial drugs. Memory immune response to meningococcal vaccine (VA-MENGOC-BC®) was observed , 12 years after its application, with anti C and B bactericidal titers of 25 and 42% respectively. We consider that these results may be influenced by systematic immunization that is carried out with this vaccine in Cuba since 1991. [ABSTRACT FROM AUTHOR]

The response of antibodies induced by the Cuban meningococcal vaccine VA-MENGOC-BC® against the strain of Neisseria meningitidis B:4:P1.19,15 was studied by the Serum Bactericidal Assay (SBA) and indirect ELISA, to measure antibodies against outer membrane vesicles (OMV) of N. meningitidis B in 184 teenagers of a polytechnical school of Ciego de Ávila who were immunized in massive campaigns 12 years before. Blood extractions were carried out before applying the first dose (T0), 4 weeks after the first dose (T1) and 4 weeks after the second dose (T2). After 12 years of this vaccination, 42% of teenagers presented bactericidal titers ≥ 1:4 against the homologous strain (B:4:P1.19,15) and 98% showed detectable antibodies against the OMVs. In the SBA, seroconversion percentage T1/T0 was 57% and T2/T0 was 60%. By ELISA, seroconversion was 59% and 54%, respectively. It was demonstrated that application of only one dose after 12 years induced an important immune response which can suggest an anamnesic response. [ABSTRACT FROM AUTHOR]

The Serum Bactericidal Assay (SBA) has been considered the "golden standard" to evaluate the serological efficacy of meningococcal vaccines, taking into account that the presence of serum bactericidal antibodies is related with protection. The levels of A and C capsular polysaccharide antibodies against these serogroups has confirmed the reliability of this assay. The SBA has also been used to asses the efficacy of serogroup B vaccines, but in some studies, the correlation between serological efficacy and protection has been poor. Ison et al developed the Whole Blood Assay (WBA) that measures the complete bactericidal activity in blood. The results obtained indicate that this model is a more sensitive marker of immunity than SBA for serogroup B. The results from the evaluation of the Cuban meningococcal vaccine (VA-MENGOC-BC®) in infants using WBA showed that after the immunisation around 50% of infants exhibited >50% lysis of the vaccine strain (B:4:P1.19,15). The results against heterologous strains were poor. Taking into account these and other results we evaluated the behavior of the WBA using lower concentrations of the bacterial suspension used as inoculum. Blood from two healthy adults was screened for bactericidal activity using the WBA, against B:4:P1.19,15(Cu 385/83) and B:15:P1.7,16 (MC58) strains. In addition to the recommended concentration (106 or 107 CFU/mL), two lower concentrations were used (105 and 104 CFU/mL). The first donor showed a lytic activity of 40% against the homologous strain, at a concentration of 107 CFU/mL. At 105 and 104 CFU/mL concentrations, lysis was around 80%. The behavior against the heterologous strain showed 25% of lysis at a concentration of 107 CFU/mL and around 60% at lower concentrations. The second donor did not show lytic activity against the homologous strain at a concentration of 107 CFU/mL, while it showed 55% and 65% lysis at 105 and 104 CFU/mL concentrations, respectively. The results against the heterologous strain were 40% of lysis for the highest concentration and 70% and 80% for concentrations of 105 and 104 CFU/mL respectively. [ABSTRACT FROM AUTHOR]

The four-component meningococcal serogroup B vaccine (4CMenB) contains antigens present in the majority of meningococci causing invasive meningococcal disease (IMD) and may potentially offer protection against strains belonging to non-B serogroups.This study aimed to evaluate the ability of 4CMenB-induced antibodies to kill, in a human serum bactericidal assay (hSBA), non-B meningococci belonging to the main genotypes responsible for IMD in Italy.Meningococci, collected between 2015 and 2017, was characterized for PorA, FetA and sequence type, and for clonal complex. Twenty non-B isolates, representative of the most frequent genotypes, were molecularly characterized for 4CMenB antigens and tested in hSBA with sera from 4CMenB-vaccinated infants and adolescents.Among twenty isolates, eleven were serogroup C, five were Y, two W and two X. All isolates contained genes encoding for fHbp and NHBA antigens and four harbored the NadA full-length encoding gene. Positive hSBA titers were obtained against all serogroup W, X and Y isolates and against five serogroup C isolates.These data show that the 4CMenB vaccine can induce bactericidal antibodies against genetically representative meningococcal W, Y and X strains from Italy. For serogroup C, different susceptibilities to killing were observed for strains with similar antigenic repertoires.

Abstract: Current concepts of vaccines against serogroup B meningococci (MenB) are mainly based on genetically variable protein antigens. Vaccine efficacy studies for meningococcal disease in developed countries are hampered by the low incidence. Licensure must therefore exclusively rely on clinical trials and laboratory investigation of meningococcal strains. In contrast to capsule polysaccharide vaccines, serum bactericidal assays for technical reasons are limited in their practicability as the surrogate of protection provided by MenB vaccines. Therefore, assays are required for reliable laboratory based assessment of expression of those specific antigen variants that are predicted to be targeted by bactericidal antibodies elicited by the vaccine. The MATS ELISA (MATS, meningococcal antigen typing system) reported recently is an example for such an assay. The paper discusses the pre- and post-licensure application of MATS, the role of reference laboratories, concepts of sustained provision of the assay, external quality assessment, and laboratory twinning. [Copyright &y& Elsevier]

Background: Given the concurrent administration of multiple vaccines during routine pediatric immunizations, efforts to elucidate the potential interference of any vaccine on the immune response to the concomitantly administered antigens are fundamental to prelicensure clinical research., Methods: This phase 3 randomized controlled trial of 13-valent pneumococcal conjugate vaccine (PCV13) versus 7-valent PCV (PCV7) assessed immune responses of concomitantly administered meningococcal group C conjugated to diphtheria toxin cross-reactive material 197 (MnCCV-CRM197) in a 2-dose infant series and 15-month toddler dose., Results: 619 subjects were randomized, 315 to PCV13 and 304 to PCV7. MnCCV-CRM197-induced immune responses were similar between the PCV13 and PCV7 groups, with >97% of the subjects achieving a ≥1:8 meningococcal serum bactericidal assay (SBA) titer after both dose 2 and the toddler dose. Geometric mean titers were lower in the PCV13 group 191.22 (167.72, 218.02) versus 266.19 (234.86, 301.71) following dose 2 and 432.28 (361.22, 517.31) versus 730.84 (642.05, 831.91) following the toddler dose. The geometric mean (GM) meningococcal SBA titer ratios (PCV13/PCV7) were 0.72 after dose 2 and 0.59 after the toddler dose. The criteria for MnCCV-CRM197 non-inferiority for GM titers were satisfied after dose 2. Percent responders was similar up to titers of 1:128. PCV13 elicited substantial antipneumococcal responses against all 13 serotypes, with ≥90% of the subjects achieving an antibody concentration ≥0.35μg/mL after dose 3 in the infant series. Safety and tolerability were similar between the vaccine groups., Conclusions: Immunogenicity results of MnCCV-CRM197 for PCV13 compared with PCV7 included lower GMTs, but the clinical significance of this is unknown as the proportion of infants achieving protective MenC antibody titers was comparable in the two groups. Percent responders were similar up to titers of 1:128. PCV13 has an acceptable safety profile in infants and toddlers, while providing expanded coverage against pneumococcal disease., (Copyright © 2013 Elsevier Ltd. All rights reserved.)