Polysaccharide Polyribosylribitol Phosphate Research Articles

Using Analytical Quality by Design to improve analytical method development in vaccines quality control: Application to an optimized quantitative high-performance anion-exchange chromatographic method

Analytical quality by design (AQbD) is an enhanced approach for the development of analytical methods. AQbD has received much industrial interest, being the subject of several recently published draft guidelines. This article demonstrates the application of AQbD to determine the quantity of non-adsorbed polysaccharide polyribosyl ribitol phosphate (PRP) and percentage of depolymerized PRP in a commercial hexavalent liquid vaccine, and establishment of an analytical control strategy (ACS). The quantification method developed is high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection, preceded by ultracentrifugation (sample preparation) for separation of the depolymerized polysaccharide from the native adsorbed polysaccharide. The first step was to develop the analytical target profile (ATP) which defines the purpose of the analytical measurement as well as the development scope. As a second step, risk assessment tools were used for identification and ranking of the critical method variables (CMVs) which have a potential impact on method performance if not controlled.Based on a multivariate Design of Experiments (DoE) approach, a proposed method operational design region (MODR) was determined for seven CMVs. Finally, the ACS was established from the understanding of the analytical method and the robustness study. This article focuses on robust and operational ranges of critical parameters linked to the ultracentrifugation and chromatographic steps for depolymerized polysaccharide content control. The design space proposed for CMVs corresponds to the ranges that ensure a product that complies with the previously established precision criteria (±2% equivalent to ± 10 % around the product criterion, which is 20 % for depolymerized polysaccharide control limit).The following design space was established from the DoE statistical modeling for ultracentrifugation critical parameters: [483,000–520,000] g for speed, [11–19]°C for temperature, [29–34] minutes for duration, and from extemporaneous to 8 min for holding time before supernatant recuperation after the ultracentrifugation. For chromatographic critical parameters, the MODR is [2–6] psi for mobile phase helium pressure, [0–7] days for mobile phase storage time, and [0–3] days for samples storage time in the autosampler at 5 °C.Methods optimized using the AQbD approach provide strong justifications during regulatory filing for the selection of analytical CMVs, and for the ACS to be applied during the lifecycle management of the method.

Chemically Synthesized Alcaligenes Lipid A as an Adjuvant to Augment Immune Responses to Haemophilus Influenzae Type B Conjugate Vaccine.

We previously identified Alcaligenes spp. as a commensal bacterium that resides in lymphoid tissues, including Peyer’s patches. We found that Alcaligenes-derived lipopolysaccharide acted as a weak agonist of Toll-like receptor four due to the unique structure of lipid A, which lies in the core of lipopolysaccharide. This feature allowed the use of chemically synthesized Alcaligenes lipid A as a safe synthetic vaccine adjuvant that induces Th17 polarization to enhance systemic IgG and respiratory IgA responses to T-cell–dependent antigens (e.g., ovalbumin and pneumococcal surface protein A) without excessive inflammation. Here, we examined the adjuvant activity of Alcaligenes lipid A on a Haemophilus influenzae B conjugate vaccine that contains capsular polysaccharide polyribosyl ribitol phosphate (PRP), a T-cell–independent antigen, conjugated with the T-cell–dependent tetanus toxoid (TT) antigen (i.e., PRP-TT). When mice were subcutaneously immunized with PRP alone or mixed with TT, Alcaligenes lipid A did not affect PRP-specific IgG production. In contrast, PRP-specific serum IgG responses were enhanced when mice were immunized with PRP-TT, but these responses were impaired in similarly immunized T-cell—deficient nude mice. Furthermore, TT-specific—but not PRP-specific—T-cell activation occurred in mice immunized with PRP-TT together with Alcaligenes lipid A. In addition, coculture with Alcaligenes lipid A promoted significant proliferation of and enhanced antibody production by B cells. Together, these findings suggest that Alcaligenes lipid A exerts an adjuvant activity on thymus-independent Hib polysaccharide antigen in the presence of a T-cell–dependent conjugate carrier antigen.

Усовершенствование процесса получения полисахарида полирибозилрибитолфосфата, используемого в производстве вакцин против гемофильной инфекции

The production of vaccines requires constant improvement of methods and tools, revision and modernization of the current technology with the aim to improve quality of the product made for the benefit of public health. The purposяe of this work was to improve the process of production of polysaccharide polyribosyl ribitol phosphate (PRP), which is the active agent of Haemophilus influenzae type b (Hib) vaccines. We investigated how PRP yield depends on the following factors: concentration of dissolved oxygen in the culture liquid, glucose concentration control method applied in cultivation, source of protein for the producer microorganism, stability of the polysaccharide at the culture liquid inactivation stage. As a result, we managed to increase the PRP yield in the culture liquid by 10%, ensured a 25% boost of the biomass accumulation rate during cultivation in the fermenter and reduced the cultivation time by 6.5 hours. The PRP loss rate at the culture liquid inactivation stage was reduced by 80%. Relying on the patented composition, we invented a new composition of the nutrient medium that meets the current regulatory requirements.

Hib Vaccines: Their Impact on Haemophilus influenzae Type b Disease.

Haemophilusinfluenzae serotype b (Hib) is an important cause of serious, invasive infections, particularly in young children. Since 1985, a series of vaccines composed of the type b capsular polysaccharide polyribosylribitol phosphate (PRP), followed by PRP conjugated to various proteins, have been licensed for use in the United States and worldwide. The conjugated vaccines offer increased immunogenicity and prolonged durability of immune protection compared to the plain PRP vaccine and increasingly are combined with other childhood vaccines for decreased cost and increased ease of vaccination. Hib vaccines have a very favorable safety profile, have been found to be either cost-saving or cost-effective by many public health agencies, and, in most countries, are initiated during early infancy as part of routine childhood immunization programs. As a result of widespread use of the vaccines, the incidence of Hib infections, and their associated morbidity and mortality, has fallen dramatically across the globe. Yet, many children remain unimmunized or underimmunized against Hib, particularly in limited-resource countries. Future efforts to further reduce the disease burden of Hib infections remain a high priority.

Vaxelis® (DTaP5-HB-IPV-Hib vaccine) as primary and booster vaccination in infants and toddlers: a profile of its use

Vaxelis® (DTaP5-HB-IPV-Hib vaccine) is a fully liquid, ready-to-use, hexavalent vaccine containing diphtheria toxoid, tetanus toxoid, five acellular pertussis antigens, hepatitis B surface antigen, inactivated poliovirus and the Haemophilus influenzae type b capsular polysaccharide polyribosylribitol phosphate conjugated to the outer membrane protein complex of Neisseria meningitidis. In pivotal clinical studies in infants and toddlers, Vaxelis® was highly immunogenic for all its component toxoids/antigens when administered by three different schedules. In general, primary endpoints of seroprotection or vaccine response rates with Vaxelis® met the predefined acceptability criteria and were noninferior to those with comparator vaccines (Infanrix® hexa or Pentacel® + Recombivax HB®). Vaxelis® can be coadministered with a number of common childhood vaccines. In clinical studies, Vaxelis® was generally well tolerated with a tolerability profile similar to that of the comparator vaccines. Available clinical data indicate that Vaxelis® is a new hexavalent vaccine option for immunization against several serious childhood infectious diseases.

PECULIARITIES OF CULTIVATION OF HAEMOPHILUS INFLUENZAE TYPE B STRAINS - PRODUCERS OF POLYRIBOSYLRIBITOL PHOSPHATE - THE MAIN COMPONENT OF POLYSACCHARIDE VACCINES

Одной из актуальных задач современной иммунобиотехнологии является разработка и внедрение эффективной вакцины против гемофильной инфекций, возбудителем которой является бактерия Haemophilus influenzae тип b (Hib). Основным действующим веществом вакцины против Hib-инфекции является капсульный полисахарид полирибозилрибитолфосфат (ПРФ), который выделяют из культуральной жидкости H. influenzae тип b. Важным технологическим этапом получения ПРФ является культивирование штамма-продуцента в условиях, позволяющих получить максимальное количество целевого продукта. На данный момент планируется подбор оптимальных условий культивирования штамма H. influenzae тип b В-7884, изолированного и идентифицированного ранее сотрудниками ФГУП «Санкт-Петербургский научно-исследовательский институт вакцин и сывороток и предприятие по производству бактерийных препаратов» ФМБА России.Цель данной работы – обобщить и проанализировать данные литературы относительно особенностей культивирования штаммов H. influenzae тип b, а также выделения основных факторов, влияющих на биосинтез ПРФ.Материалы и методы. В процессе подбора материала для написания обзорной статьи использовали базы данных Google Patents, Science Research Portal, Google Scholar, ScienceDirect, CiteSeer, Publications, ResearchIndex, Ingenta, PubMed, KEGG и др.Результаты и обсуждение. В результате анализа литературы были выделены основные факторы, влияющие на биосинтез ПРФ: природа и концентрация источников углерода и азота в составе питательной среды, концентрация факторов роста (никотиамидадениндинуклеотид, гемин, витамины), дополнительное внесение подпитки, регуляция рН в процессе культивирования, скорость перемешивания. Обобщены данные по штаммам-продуцентам ПРФ и условиям их культивирования, а также количеству синтезированного ПРФ, которое существенно зависит и от физиологических возможностей биологического агента, и от факторов, влияющих на регуляцию метаболизма.Заключение. Результаты данной работы будут учитываться при проведении исследований по оптимизации условий культивирования штамма H. influenzae тип b В-7884.

Immunogenicity and safety of a fully liquid aluminum phosphate adjuvanted Haemophilus influenzae type b PRP-CRM197-conjugate vaccine in healthy Japanese children: A phase III, randomized, observer-blind, multicenter, parallel-group study

BackgroundBroad use of monovalent Haemophilus influenzae type b (Hib) conjugate vaccines based on the capsular polysaccharide polyribosyl-ribitol phosphate (PRP), has significantly reduced invasive Hib disease burden in children worldwide, particularly in children aged <1year. In Japan, PRP conjugated to tetanus toxoid (PRP-T) vaccine has been widely used since the initiation of public funding programs followed by a routine vaccination designation in 2013. MethodsWe compared the immunogenicity and safety of PRP conjugated to a non-toxic diphtheria toxin mutant (PRP-CRM197) vaccine with the PRP-T vaccine when administered subcutaneously to healthy Japanese children in a phase III study. Additionally, we evaluated the immunogenicity and safety profiles of a diphtheria–tetanus acellular pertussis (DTaP) combination vaccine when concomitantly administered with either PRP-CRM197 or PRP-T vaccines. The primary endpoint was the “long-term seroprotection rate”, defined as the group proportion with anti-PRP antibody titers ⩾1.0μg/mL, after the primary series. ResultsLong-term seroprotection rates were 99.3% in the PRP-CRM197 group and 95.6% in the PRP-T group. The intergroup difference (PRP-CRM197 group – PRP-T group) was 3.7% (95% confidence interval: 0.099–7.336), demonstrating that PRP-CRM197 vaccine was non-inferior to PRP-T vaccine (p<0.0001). Furthermore, the “short-term seroprotection rate” (anti-PRP antibody titer ⩾0.15μg/mL) before booster vaccination was higher in the PRP-CRM197 group than in PRP-T. Concomitant administration of PRP-CRM197 vaccine with DTaP vaccine showed no differences in terms of immunogenicity compared with concomitant vaccination with PRP-T vaccine and DTaP vaccine. Although CRM197 vaccine had higher local reactogenicity, overall, both Hib vaccines had acceptable safety and tolerability profiles. ConclusionThe immunogenicity of PRP-CRM197 vaccine administered subcutaneously as a three-dose primary series in children followed by a booster vaccination 1year after the primary series induced protective levels of Hib antibodies with no safety or tolerability concerns.Clinical trial registry: Registered on ClinicalTrials.gov: NCT01379846

Immunogenicity and Efficacy of Different Haemophilus influenzae type b Vaccines

Haemophilus influenzae, a major cause of meningitis in young children leading to death and other neurological sequelae. The disease leaves 15 to 35% of the survivors with permanent disabilities, such as, mental retardation or deafness. Despite the availability of new and more powerful antibiotics children with Hib meningitis still suffer from high mortality or morbidity. The emergence of multiresistant Hib strains causes increasing difficulties in selecting proper antibiotics for the treatment. Since 1970, the capsular polysaccharide polyribosylribitol phosphate (PRP) in H. influenzae b has been the target for vaccine development. The first Hib polysaccharide vaccine licensed in 1985, proved immunogenic in human adults, but failed to elicit an immune response in children under 2 years of age who were at greatest risk of developing the invasive Hib infection. These factors led to one of the most exciting advances in pediatrics, the development of Hib conjugate vaccines. Unlike most other vaccines for preventing a particular disease which are generally similar for all types, the specific characteristics of the available Hib conjugate vaccines licensed vary from each other in structure and immunological properties. In this review the immunogenicity and efficacy of Hib vaccines including a) PRP vaccine; b) Conjugate vaccines; and c) Combination vaccines is evaluated.

Randomised controlled trial of combined diphtheria, tetanus, whole-cell pertussis vaccine administered in the same syringe and separately with Haemophilus influenzae type b vaccine at two, three and four months of age

An open randomised controlled multicentre study compared the immunogenicity and reactogenicity of three vaccines given by injection at two, three and four months of age. Children (89) received Haemophilus influenzae type b (Hib) vaccine (SmithKline Beecham Biologicals [SB]) administered in the same syringe with combined diphtheria–tetanus–whole-cell pertussis (DTP W) vaccine (Evans); 75 received Hib vaccine (SB) administered as a separate injection with DTP W vaccine; 66 received Hib vaccine (Pasteur Merieux [PM]) administered as a separate injection with DTP W vaccine. All subjects in both groups receiving Hib (SB) vaccine had levels of antibodies to the Hib polysaccharide polyribosylribitol phosphate (PRP) greater than 0.15 μg ml −1 as did 97% of those receiving Hib (PM) vaccine 1 month after administration of the final vaccine dose. Subjects in all three groups demonstrated an immunological response to pertussis, diphtheria and tetanus antigens. The geometric mean titres of the group given Hib (SB) and DTP W vaccine mixed in the same syringe were lower than the other groups. There were no apparent differences between the treatment groups in the incidence of local or systemic reactions, or serious adverse events. This study has confirmed that it is possible to halve the number of injections necessary to offer protection, with advantages to parents, children, doctors and nurses, using a combined DTP WHib vaccine and in accordance with the UK's accelerated primary immunisation schedule at two, three and four months of age.

Interchangeability of Conjugated Haemophilus influenzae Type b Vaccines in Infants

To evaluate the safety and immunogenicity of two Haemophilus influenzae type b (Hib) conjugate vaccines when administered in serial combination. These vaccines consisted of Hib capsular polysaccharide polyribosyl-ribitol phosphate (PRP) conjugated to the meningococcal outer membrane protein (OMP) complex (PRP-OMP) and H influenzae oligosaccharide conjugated to a mutant toxin (CRM197) isolated from Corynebacterium diphtheriae (HbOC). Randomized, double-blind, clinical trial evaluating five Hib vaccination regimens. Vaccine Treatment and Evaluation Units and affiliated private pediatric practices at Saint Louis (Mo) University, Vanderbilt University, Nashville, Tenn, and Baylor College of Medicine, Houston, Tex. A total of 497 healthy 2-month-old infants scheduled to receive routine immunization. Participants received either PRP-OMP or HbOC given as recommended by the manufacturer, PRP-OMP at 2 and 6 months, HbOC at 2 months, then PRP-OMP at 4 and 6 months, or PRP-OMP at 2 months and then HbOC at 4 and 6 months. Unconjugated PRP was given at 15 months to evaluate priming. Geometric mean antibody concentrations differed significantly among the groups following the second and third immunizations of the primary series and following booster immunization with unconjugated PRP. On each occasion, the groups receiving serial combinations of PRP-OMP and HbOC achieved mean antibody concentrations that equalled or exceeded those of the groups receiving a single product. Adverse reactions did not vary by group. The studied sequential combinations of Hib vaccines were safe and at least as immunogenic as either vaccine alone.

Pediatrics

As late as the 1980s, serious invasive diseases caused by<i>Haemophilus influenzae</i>type b (Hib), chiefly meningitis and epiglottitis, were important causes of morbidity and mortality in children, especially in infants younger than 15 months. The mortality associated with Hib meningitis was 3% to 6%, and permanent neurological sequelae occurred in 20% to 30% of survivors. This degree of morbidity and mortality was comparable to that observed during the last great epidemics of paralytic poliomyelitis during the 1950s. The first vaccines active against Hib disease were prepared from the Hib capsular polysaccharide polyribosylribitol phosphate (PRP) and introduced in the 1980s. These Hib PRP vaccines were immunogenic and clinically effective in children older than 18 months. However, they elicited little antibody response in infants aged 3 to 17 months, and two thirds of serious invasive Hib disease occurs in infants younger than 18 months. Despite this, in 1985 PRP vaccine administration

Measurement of anti- Haemophilus influenzae type b capsular polysaccharide antibodies by ELISA

Measurement of antibodies to the capsular polysaccharide polyribosylribitolphosphate (PRP) of Haemophilus influenzae type b by ELISA is made difficult by the poor binding of this antigen to the solid phase. Six coating conditions were compared using immune and non-immune human sera. Direct coating with PRP was inefficient. Precoating with protamine or poly- l-lysine (PLL) yielded irreproducible results and high background levels. Assays with PRP conjugated with PLL as coat were not sensitive enough. In addition, anti-PRP antibodies, especially those belonging to the IgM class, crossreacted with PLL. Coating with avidin or streptavidin followed by incubation with biotin-coupled PRP was not satisfactory either, due to binding of certain sera in the absence of PRP. Coating with PRP coupled to tyramine resulted in low backgrounds and acceptable specific binding levels. However, the finding that the binding of a few sera was only partially inhibited by soluble PRP led us to include an inhibition step in every experiment. Only optical densities inhibited by the antigen were taken into account. In view of the lack of parallelism of dilution curves from different sera, no attempt was made to express the results in weight units. They were expressed in arbitrary units calculated by comparison with internal standards. Under such conditions, the assay permitted a reproducible (interassay coefficients of variation around 10%) determination of PRP-Ab belonging to the various immunoglobulin classes and IgG subclasses and showed a good correlation with results obtained using the Farr assay.

Evidence for covalent attachment of phospholipid to the capsular polysaccharide of Haemophilus influenzae type b.

Cells of Haemophilus influenzae type b were grown in a liquid medium containing [3H]palmitate or [14C]ribose or both for two generations of exponential growth. Radiolabeled type-specific capsular polysaccharide, polyribosyl ribitol phosphate (PRP), was purified from the culture supernatant by Cetavlon precipitation, ethanol fractionation, and hydroxylapatite and Sepharose 4B chromatography. The doubly labeled ( [3H]palmitate and [14C]ribose) PRP preparation was found to coelute in a single peak from a Sepharose 4B column, suggesting that both precursors were incorporated into the purified PRP. A singly labeled ( [3H]palmitate) purified PRP preparation was found to be quantitatively immune precipitated by human serum containing antibody against PRP. The radioactivity of this preparation could not be dissociated from PRP by treatment with chloroform-methanol, 6 M urea, sodium dodecyl sulfate, or Zwittergent. Only after acid, alkaline, or phospholipase A2 treatment of PRP labeled with [3H]palmitate or [3H]palmitate and [14C]ribose followed by chloroform-methanol extraction could most of the 3H-radioactivity be recovered in the organic phase. The chloroform-soluble acid-hydrolyzed or phospholipase A2-treated product was identified as palmitic acid after thin-layer chromatography. These results strongly suggest that a phospholipid moiety is covalently associated with the H. influenzae type b polysaccharide PRP.

SAFETY AND IMMUNOGENICITY OF A NEW HAEMOPHILUS INFLUENZAE TYPE b VACCINE IN INFANTS UNDER ONE YEAR OF AGE

64 healthy infants, 2-3 months old, were randomly assigned to one of three vaccination groups which received either diphtheria pertussis-tetanus (DPT) vaccine, Haemophilus influenzaetype b capsular polysaccharide polyribosyl-ribitol phosphate (PRP) vaccine, or PRP + P (with pertussis adjuvant) vaccine in three doses at intervals of 2 months. Local and systemic reactions occurred most frequently after DPT vaccination and least frequently for PRP alone. Data for 60 infants from whom complete sera sets were available indicated that 70% of the infants who received three doses of PRP+P showed 2-fold or greater increases in titres of antibody to PRP with final values above the level assumed to give protection against invasive H. influenzae type b disease (≥0·15 μg/ml). In contrast, less than 10% of the infants who received PRP vaccine alone showed 2-fold or greater increases.