632. Clinical Experience with a New Fully Liquid Presentation of the MenACWY-CRM Vaccine. Results from Two Multicenter, Randomized, Controlled, Observer-Blind, Phase 2b Studies

Background Currently, licensed MenACWY-CRM conjugate vaccine presentation (Lyo/Liq) consists of two vials (lyophilized MenA, liquid MenCWY) to be reconstituted before injection. A new, fully liquid, single vial formulation has been developed and evaluated in two clinical studies in adolescents and adults aimed at demonstrating immunological non-inferiority of the liquid presentation for MenA. Methods Overall, 1337 subjects, 10 to 40 years of age (y), were exposed to a single 0.5 mL intramuscular dose of MenACWY Liquid and 1332 to MenACWY-CRM(Lyo/Liq). MenACWY-CRM Liquid was aged before administration, to test the vaccine immunogenicity at the end of the intended shelf-life and establish release and end of shelf life specifications. MenACWY-CRM(Lyo/Liq) was used as comparator and was not aged. In study 1 (NCT03652610), the Liquid vaccine underwent an ageing process under controlled conditions to reach ~30% MenA free saccharide (FS). In study 2 (NCT03433482), the Liquid vaccine was naturally aged at 2–8°C for approximately 24 and 30 months. Primary immunogenicity objective in both studies was non-inferiority of MenACWY-CRM liquid to licensed vaccine, as measured by human serum bactericidal assay (hSBA) geometric mean titers (GMTs) against MenA, 1-month post-vaccination. Results In both studies, for each between-group ratio of MenA hSBA GMTs, lower limits of the 95% confidence intervals (CIs) were greater than the prespecified non-inferiority margin (0.5), thus meeting the non-inferiority immunogenicity objective. Irrespectively of the vaccine presentation tested, over 82% of participants achieved MenA hSBA titers ≥ 8 in study 1 and at least 92% in study 2. The immunogenicity of MenACWY-CRM Liquid was similar to that of MenACWY-CRM(Lyo/Liq) when analyzed by serogroup, overall. No related serious adverse events were reported for both presentations. Conclusion After ageing, the new MenACWY-CRM Liquid demonstrated the ability to elicit non-inferior bactericidal responses against MenA compared to licensed formulation. The new full-liquid presentation is expected to increase the user-friendliness of the vaccine as well as to reduce reconstitution errors in the future, with a similar safety profile to that of the licensed vaccine presentation. Disclosures Javier Díez-Domingo, MD, PhD, GSK (Grant/Research Support)Sanofi (Grant/Research Support) Corinne Vandermeulen, MD PhD, GSK (Grant/Research Support)MSD (Grant/Research Support)Pfizer (Grant/Research Support) Tauseefullah Akhund, MD, MSc, MPH, GSK (Employee, Shareholder) Marco Costantini, MSc, MBA, EMPHA, GSK (Employee) Puneet Vir Singh, MBBS, PGDCD, GSK (Employee, Shareholder) Venere Basile, MSc, GSK (Employee, Shareholder) Elena Fragapane, MD, GSK (Employee, Shareholder) Maria Lattanzi, MD, PhD, GSK (Employee, Shareholder) Michele Pellegrini, MD, PhD, Michele Pellegrini (Employee, Shareholder)

1046. Immunogenicity and Safety of a Quadrivalent Meningococcal Conjugate Vaccine (MenACYW-TT) Administered as a Booster to Adults ≥ 59 Years of Age

Background MenACYW-TT (MenQuadfi®, Sanofi) is a quadrivalent (serogroups A, C, W, and Y) meningococcal tetanus toxoid conjugate vaccine. It was recently approved for use in persons aged ≥ 2 years in the US and persons aged ≥ 1 year in Europe and certain other countries; trials in infants as young as 6 weeks are ongoing. This study evaluated seroresponse after a MenACYW-TT booster given to adults who received either quadrivalent meningococcal polysaccharide vaccine (MSPV4) or MenACYW-TT three years earlier at age ≥ 56 years. Immune persistence up to 7 years after primary vaccination was also evaluated. Methods This was a Phase 3 randomized, open-label study (NCT04142242) of adults aged ≥ 59 years who participated in previous studies of MenACYW-TT vs MPSV4 (NCT01732627 and NCT02842866). The study was conducted in the US and Puerto Rico. Immune response and persistence were assessed with a serum bactericidal assay using human complement (hSBA). Sufficiency of the vaccine seroresponse was considered demonstrated if the lower limit of the 1-sided 97.5% CI for the percentage of subjects with an hSBA vaccine seroresponse against serogroups A, C, W and Y was > 40%. Safety data were collected up to 30 days after booster vaccination. Results A total of 471 persons were enrolled. Sufficiency of a MenACYW-TT booster was demonstrated for MPSV4- and for MenACYW-TT-primed subjects. hSBA seroresponse rates were higher among MenACYW-TT- vs MPSV4-primed subjects (79.3%–93.1% vs 49.2%–60.8%, respectively). Three to 7 years after primary vaccination, hSBA geometric mean titers (GMTs) and seroprotection rates (SPRs) declined in both MenACYW-TT- and MPSV4-primed subjects, with hSBA GMTs and SPRs for serogroups C, W, and Y generally remaining higher for MenACYW-TT- vs MPSV4-primed subjects; those for serogroup A were similar regardless of priming vaccine. Rates of adverse events following a MenACYW-TT booster were similar between MenACYW-TT- and MPSV4-primed subjects. No safety concerns were identified. Conclusion A MenACYW-TT booster was well tolerated and immunogenic when administered to either MPSV4- or MenACYW-primed adults aged ≥ 59 years. Up to 7 years after primary vaccination, immune persistence for serogroups C, W, and Y tended to be greater for MenACYW-TT vs MPSV4. Disclosures Corwin A. Robertson, MD, MPH, FACP, Sanofi Pasteur (Employee, Other Financial or Material Support, Stockholder) Alexandre Selmani, PhD, Sanofi Pasteur (Employee) Katherine Galarza, MD, Sanofi Pasteur (Employee) Philipp Oster, MD, Sanofi Pasteur (Employee, Stockholder)

Increasing the High Throughput of a Luminescence-Based Serum Bactericidal Assay (L-SBA)

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.

A Novel Luminescence-Based Serum Bactericidal Assay for Vibrio cholerae Reduces Assay Variation, Is Time- and Cost-Effective, and Directly Measures Continuous Titer Values

Cholera remains a significant public health burden worldwide, and better methods for monitoring cholera incidence would enhance the effectiveness of public health interventions. The serum bactericidal assay (SBA) has been used extensively for Vibrio cholerae vaccine assessments and serosurveillance. Current SBA approaches for V. cholerae rely on colony enumeration or optical density (OD600nm) readings to measure viable bacteria following complement-mediated lysis. These methods provide titer values that are constrained to discrete dilution values and rely on bacterial outgrowth, which is time consuming and prone to variation. Detection of bacterial proteins following complement-mediated lysis presents a faster and potentially less variable alternative approach independent of bacterial outgrowth. Here, we present an SBA that measures luciferase luminescence driven by lysis-released adenylate kinase. This approach is faster and less variable than growth-dependent SBAs and directly measures continuous titer values. This novel SBA method can potentially be applied to other bacteria of interest.

Endogenous complement human serum bactericidal assay (enc-hSBA) for vaccine effectiveness assessments against meningococcal serogroup B

Immunogenicity of vaccines against meningococcal serogroup B (MenB) has been assessed pre-licensure with a human serum bactericidal activity assay (hSBA), tested against small numbers of strains. We report the qualification/validation of an alternative qualitative hSBA which uses endogenous complement (enc-hSBA) present in the vaccinee’s serum. Serum samples were collected from adults pre-vaccination and post-vaccination with the 4-component MenB vaccine (4CMenB). A representative panel of invasive isolates and 4 antigen-specific indicator strains were used in qualification experiments. Each strain was tested in ≥3 experiments with pre/post-vaccination sera to evaluate intermediate precision. A 110-strain panel and the 4 indicator strains met qualification criteria, demonstrating assay precision. Assay robustness, specificity and sensitivity were demonstrated using the 4 indicator strains. Enc-hSBA is highly standardized, allows testing across large panels of epidemiologically-relevant MenB strains, and accounts for complement activity differences between vaccinees. Therefore, enc-hSBA enables a more accurate estimation of effectiveness for vaccines against MenB.

Bactericidal Activity of Serum By Brucella Abortus RB51 Outer Membrane Protein’s Combined By Brucella Abortus S99 Lipopolysaccharide Induction

Brucellosis vaccines are designed to induce the cellular immunity. An effective brucellosis vaccine is one that could induce both cellular and humoral immunity. Serum Bactericidal Assay(SBA) is an important method for the determination of vaccine humoral immunity. This study is the first to observe humoral immunity in brucellosis by SBA. Extracted B.abortus LPS and OMP’s were injected to rabbits. Group1 injected by 25µg of LPS, Group2 injected by 50µg of OMP ‘s and Group3 injected by 1ml of combined vaccine, 3 times every 2 weeks. The Groups were challenged with B.abortus 544 in the second injection. Sera were separated 2 weeks after the last injection. SBA was performed, each well was streak cultured into a plate of Brucella Agar. Colony count was done for each plate. Results have shown, the third injection of the combined vaccine, had the highest titer of 1/64, and the efficacy of the vaccine was %87.71.

Structure of a protective epitope reveals the importance of acetylation ofNeisseria meningitidisserogroup A capsular polysaccharide

Meningococcal meningitis remains a substantial cause of mortality and morbidity worldwide. Until recently, countries in the African meningitis belt were susceptible to devastating outbreaks, largely attributed to serogroup ANeisseria meningitidis(MenA). Vaccination with glycoconjugates of MenA capsular polysaccharide led to an almost complete elimination of MenA clinical cases. To understand the molecular basis of vaccine-induced protection, we generated a panel of oligosaccharide fragments of different lengths and tested them with polyclonal and monoclonal antibodies by inhibition enzyme-linked immunosorbent assay, surface plasmon resonance, and competitive human serum bactericidal assay, which is a surrogate for protection. The epitope was shown to optimize between three and six repeating units and to beO-acetylated. The molecular interactions between a protective monoclonal antibody and a MenA capsular polysaccharide fragment were further elucidated at the atomic level by saturation transfer difference NMR spectroscopy and X-ray crystallography. The epitope consists of a trisaccharide anchored to the antibody via theO- andN-acetyl moieties through either H-bonding or CH–π interactions. In silico docking showed that 3-O-acetylation of the upstream residue is essential for antibody binding, whileO-acetate could be equally accommodated at three and four positions of the other two residues. These results shed light on the mechanism of action of current MenA vaccines and provide a foundation for the rational design of improved therapies.

7. Two-Dose 4CMenB Vaccination in Adolescents Elicits a Bactericidal Activity against 15 Outbreak-Representative Meningococcal Strains

Background Meningococcal outbreaks have often been associated with N. meningitidis serogroup B (MenB) in high-income countries. We examined whether antibodies elicited by the 4-component MenB vaccine (4CMenB) in adolescents could induce complement-mediated bacterial killing of a panel of 14 genetically diverse MenB strains representative of outbreaks that occurred from 2001 to 2016 (11 from the US, 2 from the UK, and 1 from France). One N. meningitidis serogroup W (MenW) hyperendemic strain (UK, 2011) was also included in the analysis. Methods In a previous multicenter study (NCT02212457), adolescents aged 10-18y received 2 4CMenB doses 2 months apart. We tested individual sera collected from a subgroup of 20 US participants at pre-vaccination and 1 month post-second dose in a serum bactericidal assay with human complement (hSBA) against the meningococcal strain panel. Similarly, sera collected from 23 Chilean adolescents aged 11-17y (NCT00661713) were tested in a hSBA against a subset of 4 strains (3 from the US, 1 from the UK). Results At baseline, the percentage of US subjects with seroprotective titers (hSBA ≥ 1:4) ranged from 5% to 35%. One month after 4CMenB series completion, 65% to 100% had seroprotective titers (hSBA ≥ 1:4) against 11 out of the 14 MenB tested strains. The seroprotection rate was 45%, 25%, and 15% against the 3 remaining MenB strains. Against MenW, the percentage of adolescents with hSBA titers ≥ 1:4 was 15% at baseline and 95% one month after series completion. No significant changes in the percentage of subjects were observed when analysing hSBA titers ≥ 1:8. Moreover, the subset analysis indicated similar results for US and Chilean subjects for 3 out of 4 strains: the percentage of US vs Chilean subjects with hSBA titers ≥ 1:4 was 100% vs 100%; 80% vs 74%; 45% vs 52%. For the 4th strain, 65% of US subjects vs 91% of Chilean subjects showed a hSBA ≥ 1:4. Conclusion 4CMenB elicited bactericidal antibodies against a panel of 14 outbreak-representative MenB strains and 1 MenW hyperendemic strain in US adolescents. No major differences were detected in the bactericidal activity of Chilean subjects vaccinated with 4CMenB when tested against a subset of 4 MenB outbreak strains, suggesting that the immune response to 4CMenB is comparable in adolescents from different geographic areas. Disclosures Alessia Biolchi, n/a, GSK (Employee) Sara Tomei, n/a, GSK (Employee) Laura Santini, n/a, GSK (Employee) Rita La Gaetana, n/a, GSK Vaccines (Employee, Shareholder) Elena Mori, n/a, GSK (Employee) Patricia Novy, PhD, GSK (Employee, Shareholder) Rino Rappuoli, PhD, GSK (Employee) Rafik Bekkat-Berkani, M.D, GSK (Employee, Shareholder) Marzia Monica Giuliani, n/a, GSK (Employee, Shareholder) Mariagrazia Pizza, Biological Sciences, PhD, GSK Vaccines (Employee)

Intra-Laboratory Evaluation of Luminescence Based High-Throughput Serum Bactericidal Assay (L-SBA) to Determine Bactericidal Activity of Human Sera against Shigella

Despite the huge decrease in deaths caused by Shigella worldwide in recent decades, shigellosis still causes over 200,000 deaths every year. No vaccine is currently available, and the morbidity of the disease coupled with the rise of antimicrobial resistance renders the introduction of an effective vaccine extremely urgent. Although a clear immune correlate of protection against shigellosis has not yet been established, the demonstration of the bactericidal activity of antibodies induced upon vaccination may provide one means of the functionality of antibodies induced in protecting against Shigella. The method of choice to evaluate the complement-mediated functional activity of vaccine-induced antibodies is the Serum Bactericidal Assay (SBA). Here we present the development and intra-laboratory characterization of a high-throughput luminescence-based SBA (L-SBA) method, based on the detection of ATP as a proxy of surviving bacteria, to evaluate the complement-mediated killing of human sera. We demonstrated the high specificity of the assay against a homologous strain without any heterologous aspecificity detected against species-related and non-species-related strains. We assessed the linearity, repeatability and reproducibility of L-SBA on human sera. This work will guide the bactericidal activity assessment of clinical sera raised against S. sonnei. The method has the potential of being applicable with similar performances to determine the bactericidal activity of any non-clinical and clinical sera that rely on complement-mediated killing.

Safety and Immunogenicity of a Quadrivalent Meningococcal Conjugate Vaccine (MenACWY-D) in Children 9–23 Months of Age in Russian Federation: Results of Russian Part of International Study in Russia and India

Aim. Assessment of tolerability, safety and immunogenicity of the 4-valent conjugated meningococcal MenACYW-D vaccine, administered 2 times to children 1–2 years of age in the Russian Federation.Study participants. 100 children aged 9–17 months not previously vaccinated against meningococcal infection.Methods. Two doses of the MenACYW-D vaccine were administered intramuscularly at intervals of 3–6 months. Antibody titers for meningococci of serogroups A, C, W, and Y were determined using a serum bactericidal assay in the presence of human complement before the 1st vaccination and 28 days after the 2nd vaccination. Participants with titers ≥ 1:8 were considered protected from meningococcal infection caused by the corresponding serogroup of meningococci.Results. After two vaccinations, the level of seroprotection in relation to these four serogroups of meningococci was achieved in 92.9–99.0% of vaccinated children. No immediate adverse events were reported after any of the 2 doses of the vaccine studied. The frequency of local and general expected adverse reactions after any of the 2 doses of the vaccine was 45% and 40%, respectively, in terms of severity they were mostly weak and disappeared within 3 days. In general, there was no increase in the reactogenicity of the vaccine after administration of the 2nd dose compared to the 1st dose. Unexpected adverse events were recorded in 10% of the study participants, of which only one (diarrhea that stopped within one day) at the conclusion of the research physician was a causally related with the vaccine. None of the adverse events led to the early termination of participation in the study. One serious adverse event has been reported, which the physician has identified as not having a causal relationship with the vaccine being studied.Conclusions. Two dose immunization with the MenACWY-D vaccine in children 1–2 years of age in the Russian Federation was safe, well tolerated, and induced a pronounced bactericidal humoral immune response against meningococci of serogroups A, C, W, and Y.