Rabbit Complement Research Articles

High-Throughput Luminescence-Based Serum Bactericidal Assay Optimization and Characterization to Assess Human Sera Functionality Against Multiple Shigella flexneri Serotypes

Shigellosis represents a significant global health concern particularly affecting children under 5 years in low- and middle-income countries (LMICs) and is associated with stunting and antimicrobial resistance. There is a critical need for an effective vaccine offering broad protection against the different Shigella serotypes. A correlate of protection has not yet been established but there is a general consensus about the relevant role of anti-O-Antigen-specific IgG and its functionality evaluated by the Serum Bactericidal Assay (SBA). This study aims to characterize a high-throughput luminescence-based SBA (L-SBA) against seven widespread Shigella serotypes. The assay was previously developed and characterized for S. sonnei and S. flexneri 1b, 2a, and 3a and has now been refined and extended to an additional five serotypes (S. flexneri 4a, 5b, 6, X, and Y). The characterization of the assay with human sera confirmed the repeatability, intermediate precision, and linearity of the assays; both homologous and heterologous specificity were verified as well; finally, limit of detection and quantification were established for all assays. Moreover, different sources of baby rabbit complement showed to have no impact on L-SBA output. The results obtained confirm the possibility of extending the L-SBA to multiple Shigella serotypes, thus enabling analysis of the functional response induced by natural exposure to Shigella in epidemiological studies and the ability of candidate vaccines to elicit cross-functional antibodies able to kill a broad panel of prevalent Shigella serotypes in a complement-mediated fashion.

In Vitro Evaluation of Effect of Storage Time on Immunogenicity of the 10-Valent Pneumococcal Conjugate Vaccine Using Baby Rabbit Complement & HL-60 Cells

Background: The efficacy and effectiveness of a vaccine is influenced by several factors including storage duration. Additionally, vaccines may be sufficiently administered but functionality of antibody generated may be hampered with by other factors such as nutritional status of the patient. As such, one of the ways of assessing vaccine efficacy is assessing the functionality of the antibodies generated. The 10v-PCV is a highly effective vaccine used to prevent invasive pneumococcal diseases in children. However, here in Kenya, cases of pneumococcal diseases are high and challenging to treat. This study thus aimed to evaluate the effect of storage of PCV-10 storage time on the functionality of Abs generated. Methodology; An in vitro experimental research design was employed for this study. opsonophagocytic activity assay using HL-60 Cells & Baby rabbit complement was used to assess whether or not the Pneumococci Serotype IgG antibodies elicited following administration with 10v-PCV vaccine are functional. Only 10v-PCV vaccine with same production date were procured. Twenty experimental rabbits of same gender were procured and used injected intraperitoneally with 3 doses of 100µl (0.34g) of 10v-PCV vaccine after every four weeks. Antibody functionality was then assessed using the opsonophagocytic activity assay method. Results: An average of 28.9% of pneumococcus exposed to the vaccine-induced secreted antibodies were killed by the pneumococcal IgG antibody. The highest percentage of number of bacteria killed was achieved after the 3rd dose of vaccination. There was no significant influence of storage duration on opsonophagocytic activity of generated antibodies. Conclusion: Storing of 10v-PCV vaccine up to 12 weeks does not significantly affect the opsonophagocytic activity of pneumococcal IgG antibodies.

Immunogenicity of the Conjugate Meningococcal ACWY-TT Vaccine in Children and Adolescents Living with HIV.

Children and adolescents living with HIV (CALHIV) are at high risk of meningococcal infections and may present lower immune responses to vaccines. The objectives of this study were to assess the immunogenicity of the quadrivalent Men ACWY-TT vaccine (Nimenrix®) in CALHIV after a two-dose schedule and to describe possible HIV-related factors that may affect the immunogenic response. A multicenter prospective study was designed, including CALHIV followed in five hospitals in Madrid, between 2019 and 2021. Two doses of the Men ACWY-TT vaccine were administered. Serum bactericidal antibody (SBA) assays using rabbit complement (rSBA) against serogroups C, W, and Y were used to determine seroprotection and vaccine response (the proportion achieving a putative protective titer of ≥eight or a ≥four-fold rise in titer from baseline). Serum was collected at baseline, and at 3 and 12 months after vaccination. There were 29 CALHIV included, 76% of whom were perinatally infected. All were receiving TAR and presented a good immunovirological and clinical status overall. At baseline, 45% of CALHIV had seroprotective titers to at least one serogroup, with individual seroprotection rates of 24%, 28%, and 32% against C, W, and Y, respectively. After a two-dose schedule, vaccine response was 83% for each serogroup, eliciting a vaccine response to all serogroups in 69% of them. One year after vaccination, 75% of CALHIV maintained seroprotective titers against the C serogroup, and 96% against W and Y. None of the HIV-related characteristics analyzed could predict vaccine response or antibody duration. CALHIV who received effective TAR and presented a good immuno-virological situation achieved an appropriate vaccine response after two doses of the Men ACWY-TT vaccine, and antibody-mediated protection against serogroups C, W, and Y was maintained in more than 70% of the patients one year after vaccination.

Development of A Standardized Opsonophagocytosis Killing Assay for Group B Streptococcus and Assessment in an Interlaboratory Study

The placental transfer of antibodies that mediate bacterial clearance via phagocytes is likely important for protection against invasive group B Streptococcus (GBS) disease. A robust functional assay is essential to determine the immune correlates of protection and assist vaccine development. Using standard reagents, we developed and optimized an opsonophagocytic killing assay (OPKA) where dilutions of test sera were incubated with bacteria, baby rabbit complement (BRC) and differentiated HL60 cells (dHL60) for 30 min. Following overnight incubation, the surviving bacteria were enumerated and the % bacterial survival was calculated relative to serum-negative controls. A reciprocal 50% killing titer was then assigned. The minimal concentrations of anti-capsular polysaccharide (CPS) IgG required for 50% killing were 1.65–3.70 ng/mL (depending on serotype). Inhibition of killing was observed using sera absorbed with homologous CPS but not heterologous CPS, indicating specificity for anti-CPS IgG. The assay performance was examined in an interlaboratory study using residual sera from CPS-conjugate vaccine trials with international partners in the Group B Streptococcus Assay STandardisatiON (GASTON) Consortium. Strong correlations of reported titers between laboratories were observed: ST-Ia r = 0.88, ST-Ib r = 0.91, ST-II r = 0.91, ST-III r = 0.90 and ST-V r = 0.94. The OPKA is an easily transferable assay with accessible standard reagents and will be a valuable tool to assess GBS-specific antibodies in natural immunity and vaccine studies.

Distinctive features of the allelofund of blood groups of various breeds of cattle

Purpose: to study the allelofund of blood groups of cattle of different breeds.Materials and methods. 3117 Holstein cows of the black-and-white breed were studied, 848 cows of the Simmental breed, 555 cows of the Hereford breed and 172 cows of the Aberdeen-Angus breed. Blood groups were determined using a hemolysis reaction using mono-specific serum reagents, a suspension of erythrocytes and rabbit complement. Blood groups consist of alleles – a set of antigens inherited concatenated. In total, 12 systems are isolated in the blood groups of cattle. In this study, the EAV system is used, since it has the greatest variety of alternatives.Results. It was revealed that the allelofund of the Holstein black-and-white breed is represented by 44 alleles, the Simmental breed – 87 alleles, the Geford breed - 53 alleles, the Aberdeen Angus breed – 38 alleles. The most common alleles of blood groups of cattle of Holstein black-and–white breed are G2Y2'E'1Q' and I2, Simmental breed – Q', Hereford breed – b and Y1D'I', Aberdeen-Angus breed O1Q'. The frequency of occurrence of these alleles exceeds 6% and in some cases reaches 44%. It should be noted that the most common alleles of breeds remain in the leading positions in frequency of occurrence over time and are not replaced by other alleles. It is also established that in each breed there are specific alleles inherent only to it, which can be called markers of the breed. So, in the Holstein black-and–white breed, these are the alleles O1Y1G'G" and O3J'2K'O', in the Simmental – D'E'3G'I' and D'G'I'Q', in the Hereford – Y1D'I' and I2Y2G'G", in the Aberdeen-Angus – P1Y1 E'3P'2Q'G".

Safety and immunogenicity of quadrivalent meningococcal polysaccharide vaccine (MPV ACYW135) compared with quadrivalent meningococcal conjugate vaccine (Menactra®) in Malian children

ABSTRACT Affordable, polyvalent meningococcal vaccines are needed for use in emergency reactive immunization campaigns. A phase IV randomized, observer-blind, controlled study compared the safety and immunogenicity of a quadrivalent meningococcal polysaccharide vaccine (MPV-4, MPV ACYW135) and quadrivalent meningococcal ACWY conjugate vaccine (MCV-4, Menactra®). Healthy, 2- to 10-year-old children in Bamako, Mali, were randomized 1:1 to receive one dose of MPV-4 or MCV-4. Safety outcomes were evaluated for 6 months post-immunization. Immunogenicity for all serogroups was assessed for non-inferiority between MPV-4 and MCV-4 30 days post immunization by serum bactericidal antibody assay using baby rabbit complement (rSBA). From December 2020 to July 2021, 260 healthy subjects were consented and randomized. At Day 30 post-immunization, the proportions of subjects with rSBA titers ≥ 128 for all serogroups in the MPV-4 group were non-inferior to those in MCV-4 group. The proportions of subjects with rSBA ≥ 4-fold increase and rSBA titers ≥ 8 for all serogroups were similar among vaccine groups (P > .05). Geometric Mean Titers and Geometric Mean Fold Increases for all serogroups in both vaccine groups were similar (P > .05). Few local and systemic post-immunization reactions of similar severity and duration were observed within 7 days and were similar in both groups (P > .05). All resolved without sequelae. Unsolicited adverse events were similar in both groups regarding relationship to study vaccine, severity and duration. No serious adverse events were reported during the study period. MPV ACYW135 showed a non-inferior immunogenicity profile and a comparable reactogenicity profile to MCV-4 in Malian children aged 2–10 years. Clinical Trial Registration: NCT04450498.

Evaluation of Monkeypox- and Vaccinia virus-neutralizing antibodies in human serum samples after vaccination and natural infection.

In early to mid-2022, an unexpected outbreak of Monkeypox virus infections occurred outside the African endemic regions. Vaccines originally developed in the past to protect against smallpox are one of the available countermeasures to prevent and protect against Orthopoxvirus infections. To date, there are few studies on the cross-reactivity of neutralizing antibodies elicited by previous vaccinia virus-based vaccination and/or Monkeypox virus infection. The aim of this study was to evaluate a possible approach to performing Monkeypox and vaccinia live-virus microneutralization assays in which the read-out is based on the production of cytopathic effect in the cell monolayer. Given the complexity of Orthopoxviruses, the microneutralization assay was performed in such a way as to uncover a potential role of complement, with and without the addition of an external source of Baby Rabbit Complement. A set of human serum samples from individuals who had been naturally infected with Monkeypox virus and individuals who may have and not have undergone vaccinia virus vaccinations, was used to evaluate the performance, sensitivity, and specificity of the assay. The results of the present study confirm the presence and cross-reactivity of antibodies elicited by vaccinia-based vaccines, which proved able to neutralize the Monkeypox virus in the presence of an external source of complement.

New Complement Dependent Cytotoxicity Assay and its application to COVID-19 patient sera harboring anti-lymphocyte antibodies.

Abstract Complement dependent cytotoxicity (CDC) is one of the ways IgM and IgG antibodies can mediate the lysis of target cells, being IgM the antibody (Ab) isotype with the highest CDC activity. In a cohort of hospitalized COVID-19 patients we identified that 27% carried IgM anti-lymphocyte Ab in their sera. To quantify the sera potential lytic activity against healthy donor (HD) PBMC we developed a flow-based CDC assay with an internal negative control. The assay allows for normalization of the results and therefore inter-experiment data comparison without the need of using counting beads. In short, after CFSE labelling, 2 million PBMC are incubated with either complete media (CM) or with the different experimental conditions (Ab, sera, plasma), then they are washed thoroughly before adding them to equal numbers of unlabeled PBMC (internal negative control). This mixture of hot CFSE+ and cold CFSE-targets is then incubated with rabbit complement for an hour, stained and flow. A “ratio tube” containing the mixture with the CM incubated CFSE cells, is left without rabbit complement at 4 degrees and used to calculate the initial CFSE+/CFSE− ratio. We calculate the percentage of CDC lysis based on the CFSE+/CFSE− ratio in each tube after normalizing it to the ratio tube. Deploying this assay, we found CDC activity against B and T cells in five and two COVID-19 patients’ sera, respectively, out of 23 patients tested. This improved CDC assay shows high reproducibility and allows for quick, reliable, and easy test of multiple conditions. This work was supported by the Divisions of Intramural Research of NIAID, NIH

Testing of a novel liposomal anti-Pseudomonasvaccine based on outer-membrane protein F (OprF) of Pseudomonas aeruginosa

Abstract Pseudomonas aeruginosa is an opportunistic pathogen associated with an increased risk of morbidity and mortality among cystic fibrosis patients and immunocompromised individuals. Current antibiotics in the market are not successful against P. aeruginosa infections due to the pathogen’s ability to cause antibiotic resistance. Therefore, our prime objective is to formulate a liposomal-based anti-Pseudomonas vaccine that can produce long-lasting IgG antibodies and T cell memory. The vaccine we introduced accommodates several B cell epitopes of the outer-membrane protein F (OprF) of P. aeruginosa. The recombinant OprF antigen was conjugated with a Toll-like receptor 2/1 (TH1/TH2) agonist, Pam 3CysSK 4, and formulated into dipalmitoylphosphatidylcholine (DPPC)/cholesterol (Chol) liposomes. Our results show that the resulting vaccine conjugate can successfully produce high anti-OprF antibody titers in mice. These antibodies can also recognize intact P. aeruginosa bacteria and kill them with the support of rabbit complement and murine macrophage RAW264.7 cells. To further enhance the vaccine efficacy, the liposome was optimized with 2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycerol-3-phospho-(1′-rac-glycerol) (DMPG), Chol, Pam 3CysSK 4and Quillaja Saponaria-derived saponin adjuvant QS21. The mice immunized with the modified vaccine elicit higher antibody titers and increased IgG 2a antibodies. Overall, our liposomal vaccines were found to be highly effective at generating a balanced and robust TH1/TH2 response. Supported by grants from NIH (R01AI148570)

Synthesis and Evaluation of a Self-Adjuvanting Pseudomonal Vaccine Based on Major Outer Membrane Porin OprF Epitopes Formulated with Low-Toxicity QS-21-Containing Liposomes.

Pseudomonas aeruginosa (PA) is a Gram-negative pathogen that the World Health Organization has ranked as a priority 1 (critical) threat. One potential prophylactic approach to preventing or reducing the incidence of PA would be development of a long sought-after vaccine. Both antibody and CD4+ T-cell responses have been noted as playing key roles in protection against infection. In these studies, we have designed a prototype vaccine consisting of several known linear B-cell epitopes derived from an outer membrane porin F (OprF). The resulting thiol-containing protein was conjugated to a version of the lipopeptide-based Toll-like receptor agonist Pam3CysSK4Mal (10) containing a maleimide moiety and formulated into dipalmitoylphosphatidylcholine (DPPC)/cholesterol (Chol) liposomes. Mice immunized with the resulting vaccine generated antibodies that bound PA14 (serotype O10) in vitro and induced opsonization in the presence of rabbit complement and murine macrophage RAW264.7 cells. The liposome was optimized to contain 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DMPG), Chol, Pam3CysSK4-OprF (12) and the Quillaja saponaria-derived saponin adjuvant QS-21. The resulting vaccine formulation produced significantly higher antibody titers, increased the IgG2a antibody isotype, and increased the number of IgG-producing B-cells as well as splenic primed T-cells. In summary, the liposomal vaccine platform was found highly useful for the generation of a robust and balanced TH1/TH2 response.

In Memoriam: Soldano Ferrone MD, PhD (1940–2023)

In Memoriam: Soldano Ferrone MD, PhD (1940–2023)

Identification of a Novel Keto Sugar Component in Streptococcus pneumoniae Serotype 12F Capsular Polysaccharide and Impact on Vaccine Immunogenicity.

Implementation of conjugate vaccine technology revolutionized the ability to effectively elicit long-lasting immune responses to bacterial capsular polysaccharides. Although expansion of conjugate vaccine serotype coverage is designed to target residual disease burden to pneumococcal serotypes not contained in earlier vaccine versions, details of polysaccharide Ag structure, heterogeneity, and epitope structure components contributing to vaccine-mediated immunity are not always clear. Analysis of Streptococcus pneumoniae serotype 12F polysaccharide by two-dimensional nuclear magnetic resonance spectroscopy and mass spectrometry revealed a partial substitution of N-acetyl-galactosamine by the keto sugar 2-acetamido-2,6-dideoxy-xylo-hexos-4-ulose (Sug) in up to 25% of the repeat units. This substitution was not described in previous published structures for 12F. Screening a series of contemporary 12F strains isolated from humans (n = 17) identified Sug incorporation at varying levels in all strains examined. Thus, partial Sug substitution in S. pneumoniae serotype 12F may have always been present but is now detectable by state-of-the-art analytical techniques. During the steps of conjugation, the serotype 12F Sug epitope is modified by reduction, and both polysaccharide PPSV23 and conjugate PCV20 vaccines contain 12F Ags with little to no Sug epitope. Both PCV20 and PPSV23 vaccines were evaluated for protection against circulating 12F strains with varying amounts of Sug in their repeat unit based on an opsonophagocytic killing assay involving HL-60 cells and rabbit complement. Both vaccines elicited human-derived neutralizing Abs against serotype 12F, independent of Sug level between ∼2 and 25 mol%. These findings suggest that the newly identified serotype 12F Sug epitope is likely not an essential epitope for vaccine-elicited protection.

Immunogenicity and Safety of a Quadrivalent Meningococcal Tetanus Toxoid­-Conjugate Vaccine (MenACYW­-TT) Administered Concomitantly with Pneumococcal Conjugate Vaccine in Healthy Toddlers in the Russian Federation: a Phase III Randomized Study

Relevance. Invasive meningococcal disease (IMD) has high morbidity and mortality, with infants and young children among those at greatest risk.Materials & Methods. A phase III, open-­label, randomized study in toddlers aged 12–23 months evaluated the immunogenicity and safety of MenACYW­TT, a tetanus toxoid conjugated vaccine against meningococcal serogroups A, C, W, and Y, when coadministered with paediatric vaccines (measles, mumps and rubella [MMR]; varicella [V] in South Korea and Thailand; 6­in­1 combination vaccine against diphtheria, tetanus, pertussis, polio, hepatitis B and Haemophilus influenzae type b [DTaP­IPVHepB­Hib] in Mexico and pneumococcal conjugate vaccine [PCV13]) in the Russian Federation (NCT03205371). This manuscript reports the outcome of the part of the study conducted in the Russian Federation using PCV13 as the co­administered vaccine. Immunogenicity to each meningococcal serogroup was assessed by serum bactericidal antibody assay using human complement (hSBA) and, for a subset of subjects, baby rabbit complement (rSBA). Vaccine safety profiles were described up to 30 days postvaccination.Results. A total of 1,183 participants were enrolled in the study, out of which 400 were from the Russian Federation. The proportion with seroprotection (hSBA ≥1:8) to each meningococcal serogroup at Day 30 was comparable between the MenACYW­-TT and MenACYW-­TT + PCV13 groups (≥91% and ≥84%, respectively). The safety profiles of MenACYW­-TT and PCV13, when given alone or concomitantly, were generally comparable.Conclusion. Coadministration of MenACYW­-TT with pneumococcal conjugate vaccine in toddlers had no clinically relevant effect on the immunogenicity and safety of any of the vaccines.

A phase 3, randomized, controlled, open-label study to evaluate the persistence up to 5 years of 1 or 2 doses of meningococcal conjugate vaccine MenACWY-TT given with or without 13-valent pneumococcal conjugate vaccine in 12–14-month-old children

BackgroundImmunogenicity and safety up to 5 years after administration of 1 or 2 doses of quadrivalent meningococcal serogroup A, C, W, and Y tetanus toxoid conjugate vaccine (MenACWY-TT) given alone or with 13-valent pneumococcal conjugate vaccine (PCV13) in children was investigated. MethodsThis phase 3 study randomized healthy 12–24-month-olds to MenACWY-TT at Month 0 (ACWY1d), MenACWY-TT at Months 0 and 2 (ACWY2d), MenACWY-TT and PCV13 at Month 0 (Co-Ad), or PCV13 at Month 0 and MenACWY-TT at Month 2 (PCV13/ACWY). Immune responses 1, 3, and 5 years after primary vaccination were evaluated with serum bactericidal activity using rabbit complement (rSBA) titers ≥ 1:8 and geometric mean titers (GMTs). Evaluation of serious adverse events up to 5 years after primary vaccination are reported. ResultsOf the 802 children randomized in the study, 619 completed the study through Year 5. Immune responses after vaccination declined over time but were higher 5 years after vaccination compared with levels before vaccination. At Year 5, the percentages of children with rSBA titers ≥ 1:8 across all serogroups were 20.5 %−58.6 %, 28.4 %−65.8 %, 23.9 %−52.8 %, and 19.4 %−55.8 % in the ACWY1d, ACWY2d, Co-Ad, and PCV13/ACWY groups, respectively. Comparable antibody persistence at Year 5 was observed for participants receiving 1 or 2 doses of MenACWY-TT, although GMTs were elevated in those who received 2 versus 1 dose. The percentage of children with protective antibody titers at Year 5 was similar in participants who received PCV13 and MenACWY-TT compared with that observed for participants who only received 1 or 2 MenACWY-TT doses. No new safety concerns were identified during the study period. ConclusionAntibody responses persisted in the majority of children up to 5 years after primary vaccination with MenACWY-TT administered in a 1- or 2-dose regimen with or without PCV13, with no new safety concerns identified.ClinicalTrials.gov Identifier NCT01939158; EudraCT number 2013–001083-28.

Evaluation of Meningococcal Serogroup C Bactericidal Antibodies after Primary Vaccination: A Multicentre Study, Italy.

Here, we evaluated over time in different cohorts of children vaccinated against serogroup C Neisseria meningitidis, the presence of antibodies with neutralizing activity. A total of 348 sera samples of enrolled children by year since vaccination (<1 year- up to 5 years), starting from February 2016 to December 2017, were collected in three collaborating centers. Meningococcal serogroup C (MenC) antibody titers were measured with a serum bactericidal antibody (SBA) assay using rabbit complement (rSBA) following standard operating procedures. The cut-off of rSBA titer ≥ 8 is considered the correlate of protection. We observed a significantly declining of bactericidal rSBA titers by 23% every year, for every 1-year from vaccination (Adjusted PR = 0.77, 95% CI: 0.71–0.84). The proportions of children with bactericidal antibodies, immunized with the meningococcal serogroup C conjugate (MCC) vaccine, declined from 67.7% (95% CI: 48.6–83.3%) one year after vaccination, to 36.7% (95% CI: 19.9–56.1%) five years after vaccination (chi-square for linear trend, p < 0.001). Children vaccinated with the tetravalent meningococcal serogroup ACWY vaccine resulted in a high proportion of bactericidal rSBA MenC titer ≥ 1:8 (90.6%, 95% CI: 79.3–96.9%) after a mean time of seven months. Overall, the results provide some evidences on the evaluation of meningococcal serogroup C bactericidal antibodies after primary vaccination.

Comparing the meningococcal serogroup C immune response elicited by a tetanus toxoid conjugate quadrivalent meningococcal vaccine (MenACYW-TT) versus a quadrivalent or monovalent C tetanus toxoid conjugate meningococcal vaccine in healthy meningococcal vaccine-naïve toddlers: A randomised, controlled trial

ABSTRACT MenACYW-TT (MenQuadfi®) is a quadrivalent meningococcal tetanus toxoid conjugate vaccine licensed in Europe for use in individuals ≥12 months. This study assessed whether serogroup C immune responses with MenACYW-TT were at least non-inferior, or superior, to those of quadrivalent meningococcal ACWY (MCV4-TT; Nimenrix®) and monovalent meningococcal C (MenC-TT; NeisVac-C®) vaccines in toddlers (12–23 months). In this modified, double-blind Phase III study (NCT03890367), 701 toddlers received one dose of MenACYW-TT (n = 230), MCV4-TT (n = 232) or MenC-TT (n = 239). Serum bactericidal assays with human (hSBA) and baby rabbit (rSBA) complement were used to measure anti-meningococcal serogroup C antibodies at baseline and 30 days post-vaccination. A sequential statistical approach was used for primary and secondary objectives. For the primary objectives, superiority of serogroup C was assessed in terms of hSBA seroprotection rates (defined as titers ≥1:8) and GMTs for MenACYW-TT compared to MCV4-TT, and rSBA GMTs compared to MenC-TT. The safety of all vaccines within 30 days post-vaccination was described. When administered as a single dose to meningococcal vaccine-naïve healthy toddlers the superiority of the MenACYW-TT serogroup C immune response versus MCV4-TT was demonstrated for hSBA GMTs (ratio 16.3 [12.7–21.0]) and seroprotection (difference 10.43% [5.68–16.20]); and versus MenC-TT in terms of rSBA GMTs (ratio 1.32 [1.06–1.64]). The safety profiles of a single dose of MenACYW-TT, MCV4-TT and MenC-TT were similar. In meningococcal vaccine-naïve toddlers, MenACYW-TT induced superior immune responses to serogroup C versus MCV4-TT in terms of hSBA seroprotection and GMTs and versus MenC-TT in terms of rSBA GMTs.

Safety and immunogenicity of an investigational quadrivalent meningococcal tetanus toxoid conjugate vaccine (MenACYW-TT) co-administered with routine pediatric vaccines in infants and toddlers: A Phase II study

BackgroundThe MenACYW-TT conjugate vaccine is approved for prevention of invasive meningococcal disease (IMD) as a single dose in individuals ≥2 years of age in the United States and ≥12 months in EU and some other countries. This Phase II study evaluated the safety and immunogenicity of this vaccine and of concomitant pediatric vaccines in infants/toddlers (6 weeks–15 months of age). MethodsFive schedules of the MenACYW-TT conjugate vaccine were evaluated in the United States: 2, 4, 6, and 12 months; 2, 4, 6, and 15 months; 2, 4, and 12 months; 6 and 12 months; and 12 months alone. Routine pediatric vaccines (DTaP-IPV/Hib, PCV7/PCV13, MMR, and varicella) were administered per approved schedules. Proportions of participants with serum bactericidal antibodyassay with human complement (hSBA) titers ≥1:4 and ≥1:8, SBA with baby rabbit complement (rSBA) titers ≥1:8 and ≥1:128, and immune responses against concomitant vaccines were determined. ResultsTenderness and irritability were the most frequent solicited injection site and systemic reactions. Similar proportions of participants achieved an hSBA titer ≥1:8 for all four serogroups regardless of whether 2 or 3 doses were administered in the first year of life. Following a second-year dose, 91–100% of participants achieved the threshold for all 4 serogroups in all schedules regardless of the number of doses in the first year of life. Similar responses were seen with rSBA. Immunogenicity and safety profile of concomitant vaccines was similar whether the MenACYW-TT conjugate vaccine was administered or not. ConclusionMenACYW-TT conjugate vaccine administered with pediatric vaccines is safe and immunogenic regardless of the schedule and does not affect the immunogenicity or safety of the concomitant vaccines. Clinical trial registryNCT01049035.

03. Immunogenicity and Safety of a Quadrivalent Meningococcal Conjugate Vaccine (MenACYW-TT) Administered as a Booster Dose in Adults and Adolescents Vaccinated Against Meningococcal Disease 3 - 6 Years Earlier

BackgroundBooster doses of meningococcal conjugate vaccines may induce long-term protection against invasive meningococcal disease. MenACYW-TT [MenQuadfi®] is a quadrivalent meningococcal conjugate vaccine, licensed for use in ages 2 years and older in USA. The vaccine is also licensed in ages 12 months and older in EU and other countries.MethodsA phase IIIb study (NCT04084769) was conducted to evaluate the persistence of immune response in adults and adolescents primed 3-6 years earlier with either MenACYW-TT or MCV4-CRM (Menveo®) and, safety and immunogenicity of MenACYW-TT when administered as a booster dose with or without concomitant administration with MenB vaccines (Bexsero® and Trumenba®). Serum bactericidal assays with human complement (hSBA) and baby rabbit complement (rSBA) were used to measure antibodies against vaccine serogroups at baseline (Day 0 [D0]), D06 (in a subset) and 30 days post-vaccination (D30). Safety data were collected up to 6 months post-vaccination.ResultsAt D0, the GMTs were higher in subjects primed with MenACYW-TT vs MCV4-CRM for serogroups C, Y and W, and were comparable for serogroup A. At D0, all hSBA GMTs were higher than those observed pre-priming dose, suggesting persistence of immunity. Sufficiency of hSBA seroresponse ( >75%) was demonstrated following administration of MenACYW-TT booster dose regardless of the priming vaccine administered 3-6 years earlier. Vaccine seroresponse in a subset of participants at D06 ranged from 77.8% (95%CI 62.9%; 88.8%) for serogroup A to 97.8% (88.5%; 99.9%) for serogroup W suggesting a quick onset of immune response post-booster. Post-vaccination (D30) hSBA GMTs were comparable for serogroups A, Y and W regardless of the nature of the priming vaccine and were higher for serogroup C in subjects primed with MenACYW-TT vaccine. The MenACYW-TT booster dose was well-tolerated and had similar safety profiles regardless of the priming vaccine. The safety profiles were comparable regardless of the MenB vaccine co-administered with MenACYW-TT vaccine.ConclusionMenACYW-TT used as priming vaccine was able to demonstrate persistence of immune response 3-6 years later. MenACYW-TT elicits robust booster responses in adults and adolescents primed with MenACYW-TT or MCV4-CRMDisclosures Betzana Zambrano, MD, Sanofi Pasteur (Employee) Germán Áñez, MD, Sanofi Pasteur (Other Financial or Material Support, Former employee) Sue Jiayuan, MSc, Sanofi Pasteur (Independent Contractor) Judy Pan, PhD, Sanofi Pasteur (Employee) Habiba Arroum, MD, Sanofi Pasteur (Employee) Kucku Varghese, PhD, Sanofi Pasteur (Employee) Emilia Jordanov, MD, Sanofi Pasteur (Employee, Shareholder) Mandeep S. Dhingra, MD, Sanofi Pasteur (Employee, Shareholder)

Functional Similarity of Proposed Biosimilar ABP 798 with Rituximab

▪BackgroundRegulatory guidelines governing the development of biosimilars require that proposed biosimilars undergo comprehensive structural and functional characterization prior to the initiation of clinical investigation. ABP 798 is being developed as a biosimilar to rituximab. The reference product is approved in the US for treatment of non-Hodgkin's lymphoma, chronic lymphocytic leukemia, severe rheumatoid arthritis, granulomatosis with polyangiitis, and microscopic polyangiitis. Development of biosimilars is challenging as even small variations in structure have the potential to alter the function of the molecule and thus may impact efficacy, safety, and/or bioavailability. Here we compare ABP 798 and rituximab reference product with respect to binding properties (CD20, C1q, FcRn, and Fcγ receptors), antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and induction of apoptosis. The reference product was sourced from the US (rituximab US) and the EU (rituximab EU).MethodsBinding of ABP 798 and rituximab to the CD20 antigen was characterized using a cell-based CD20 binding assay utilizing the CD20 expressing WIL2-S human B-lymphoblastoid cell line. A direct binding ELISA was used to assess the binding of the Fc domain of ABP 798 to C1q. Binding of the Fc moiety of ABP 798 and rituximab to FcγRIa, FcγRIIa, FcγRIIb, and FcγRIIIa (158V and 158F) were evaluated in AlphaLISAÒ competitive binding assays. Binding to FcRn was evaluated by an AlphaScreenÒ competitive binding assay. ADCC activity was evaluated in a functional cell-based assay, with WIL2-S cells used as target cells and NK92-MI cells, stably transfected with human CD16 (FcγRIIIa [158V]), used as effector cells. CDC activity was evaluated in a functional cell-based assay using the WIL2-S cell line and baby rabbit complement. Induction of apoptosis was assessed by measuring activation of caspase 3/7 in SU-DHL-4 cells, a CD20 expressing human B cell lymphoma cell line.ResultsRelative binding (%) as compared to an ABP 798 reference standard was similar between ABP 798 and rituximab (Table 1).The dose response profiles and relative activity for ADCC and CDC were also similar (ADCC relative cytotoxicity: ABP 798, 88%; rituximab US, 85%; rituximab EU, 86%; CDC relative cytotoxicity: ABP 798, 103%; rituximab US, 102%; rituximab EU, 104%). The dose-response profile for induction of caspase 3/7 was similar between ABP 798 and rituximab as well.ConclusionsThe results presented here demonstrate that ABP 798 is similar to rituximab sourced from both US and EU in terms of biological activity across the range of tested binding and functional assays. The functional assays tested the major and minor mechanisms of action related to clinical efficacy across approved rituximab indications and these results provide a foundation for further clinical development of ABP 798. [Display omitted] DisclosuresMcBride:amgen: Employment; amgen: Other: stockholder. Maher:amgen: Employment; amgen: Other: stockholder. Sweet:amgen: Employment; amgen: Other: stockholder. Foltz:amgen: Employment; amgen: Other: stockholder. Kuhns:amgen: Other: stockholder; amgen: Employment.

Immunogenicity and safety of an investigational quadrivalent meningococcal conjugate vaccine administered as a booster dose in children vaccinated against meningococcal disease 3 years earlier as toddlers: A Phase III, open-label, multi-center study

ABSTRACT Booster doses of meningococcal conjugate vaccines induce long-term protection against invasive meningococcal disease. We evaluated the immunogenicity and safety of a booster dose of MenACYW-TT in pre-school children who were primed 3 years earlier with MenACYW-TT or MCV4-TT (Nimenrix®). In this Phase III, open-label, multi-center study (NCT03476135), children (4–5 years old), who received a primary dose of MenACYW-TT or MCV4-TT as toddlers in a previous study, received a booster dose of MenACYW-TT. Titers of antibody against meningococcal serogroups A, C, W and Y were measured by serum bactericidal assay using human (hSBA) and baby rabbit (rSBA) complement in samples collected before (D0) and 30 days after (D30) booster vaccination. Safety was assessed over the 30-day study period. Ninety-one participants received the booster dose. In both study groups, hSBA titers increased from D0 to D30; serogroup C titers [95% confidence interval] were higher in the MenACYW-TT-primed vs MCV4-TT-primed group at D0 (106 [73.2, 153] vs 11.7 [7.03, 19.4], respectively) and D30 (5894 [4325, 8031] vs 1592 [1165, 2174], respectively); rSBA results were similar. Nearly all participants achieved ≥1:8 hSBA and rSBA titers at D30, which were higher or comparable to those observed post-primary dose, suggesting rapid booster responses. At D0, all hSBA and rSBA titers were higher than those observed pre-primary dose, suggesting persistence of immunogenicity. The MenACYW-TT booster dose was well-tolerated and had similar safety outcomes across study groups. These findings suggest that MenACYW-TT elicits robust booster responses in children primed 3 years earlier with MenACYW-TT or MCV4-TT.