Development of a high-resolution magic-angle spinning nuclear magnetic resonance identity assay of the capsular polysaccharide from Haemophilus influenzae type b present in cetavlon precipitate

We describe the use of high-resolution magic-angle spinning nuclear magnetic resonance to control the identity of the capsular polysaccharide from Haemophilus influenzae type b (Hib) present in the cetavlon precipitate. This step is one of the earliest in the purification of this polysaccharide, which is further used in the production of Hib polysaccharide-protein conjugate vaccine. The effects of sample procedure and magnetic field strength have been investigated. Since this assay is rapid and simple, it may represent a useful technique for characterization of polysaccharides present in complex and insoluble matrices. Moreover, it allows a rapid evaluation of the structure of the produced polysaccharides very early on during the production process and is as such an essential analytical tool before starting the purification process.     

Presence of multiple copies of capsulation loci in invasive Haemophilus influenzae type b (Hib) strains in Japan before introduction of the Hib conjugate vaccine

Despite the effectiveness of the Hib vaccine, multiple amplification of the capb locus contributes to vaccine failure. However, there has been no report on the effect of Hib locus amplification in Japan. We examined 24 Hib strains from Japanese children with invasive diseases due to Hib. Although all strains showed the same capb sequence, Southern blot analysis showed that four strains (16.7%) harbored multiple copies (more than two) of the capb locus. Careful analysis of the locus in circulating Hib strains is necessary now that the Hib vaccine has been introduced into Japan.     

Recent trends in pediatric Haemophilus influenzae type B infections in Canada. Immunization Monitoring Program,Active (IMPACT) of the Canadian Paediatric Society and the Laboratory Centre for Disease Control.

OBJECTIVE: To describe changes in the number of cases of Haemophilus influenzae type b (Hib) infections among Canadian children before and after the introductory phases of Hib vaccination. DESIGN: Multicentre case series. SETTING: All 10 pediatric tertiary care centres across Canada participating in the Immunization Monitoring Program, Active (IMPACT) of the Canadian Paediatric Society and the Laboratory Centre for Disease control. PATIENTS: Children with a Hib infection admitted to any of the participating hospitals from 1985 to 1994. Annual case totals from 1985 to 1990 were determined from records of hospital laboratories or coded discharge diagnoses, or both. From 1991 to 1994 intensive case surveillance was conducted on the wards in addition to thorough record searches as above. OUTCOME MEASURES: Estimated annual case totals for 1985-90. For 1991-94 intensive surveillance for quarterly case totals, yearly age distribution of cases, and proportion of recent cases that represent vaccination failures or missed opportunities to prevent infection. RESULTS: The total number of Hib cases from 1985 to 1990 was 2095; from 1991 to 1994, there were 326 laboratory-confirmed cases and 15 probably cases supported by Hib antigen detection. The annual number of cases declined from an estimated 485 in 1985 to 24 in 1994, a decrease of 95.1%. The steepest interannual decrease (63.7%) occurred between 1992 and 1993, following the introduction of infant-based vaccination programs across Canada. The number of Hib cases involving children most at risk (those 6 to 18 months old) decreased from 78 in 1991 to 4 in 1994. Of the 24 cases in 1994, 6 were categorized as preventable, 1 was fatal, and 8 were vaccine failures (2 of which involved currently used vaccines). CONCLUSION: The prevalence of Hib infections reported by the IMPACT centres has declined greatly since the introduction of vaccination programs. However, deaths and complications continue to occur, attesting to the need to vaccinate all eligible infants and children against this virulent pathogen.     

A novel Enzyme-Linked Immuno-Sorbent Assay (ELISA) for the quantification of total and free polysaccharide in Haemophilus influenzae b–Tetanus toxoid conjugate vaccines in monovalent and combined vaccine formulations

Current Haemophilus influenzae b conjugate vaccines (Hib), which are made of purified capsular polysaccharide (poly-ribosyl-ribitol-phosphate; PRP) conjugated to a carrier protein, are almost completely evaluated by physico-chemical methods to ensure the integrity and stability of the vaccine and consistency of manufacture of batches. The absence of a potency assay makes the quantification of total PRP content (in SI units) and of % free polysaccharide in final fills or bulk components of Hib vaccines critical release tests for both manufacturers and national control authorities. Here we describe a simple and sensitive Enzyme-Linked Immuno-sorbent Assay (ELISA) which has been developed to quantify total and free PRP content in Hib–TT vaccine alone or when in combination with other vaccines. The assay is robust, specific and highly sensitive.     

Naturally acquired immunity to Haemophilus influenzae type B in healthy Cuban children

We have evaluated the prevalence of antibody to immunogenicity of Haemophilus influenzae type b (Hib) in a group of 4 to 5 years old healthy children, who were too old to be included in the first vaccinated cohort when Hib vaccination begun in Cuba in 1999. Serum capsular polysaccharide specific IgG antibody concentrations were measured in 974 healthy children, between February and May 2002. The prevalence of Hib nasopharyngeal carriage was also estimated. The majority of children (99.7%) had more than 1 microg/ml of antibody. The preliminary report of the nasopharyngeal cultures was positive for H. influenzae in 16 children, but in only one was confirmed as Hib after serotyping (0.1% Hib nasopharyngeal carrier). These results provide evidence that in Cuba the natural active immunity to Hib can be acquired at an early age.     

Trends in Haemophilus influenzae type b infections in adults in England and Wales: surveillance study

Objective To describe invasive Haemophilus influenzae type b (Hib) infections in individuals aged 15 years or older in England and Wales between 1991 and 2003.Design Prospective, laboratory based surveillance of invasive Hib infections and cross sectional seroprevalence study.Setting England and Wales.Participants Cases were confirmed by isolation of H influenzae from a normally sterile site, or from a non-sterile site in cases with a diagnosis of epiglottitis. Excess serum samples collected from English 30-39 year olds as part of a national serosurvey were identified for the years 1990, 1994, 1997, 2000, and 2002.Main outcome measures The number of invasive Hib infections from 1991 to 2003. Population immunity to H influenzae type b in English adults was also measured.Results After routine infant immunisation was introduced in October 1992, adult Hib infections decreased initially but then rose from a low in 1998 to reach prevaccine levels in 2003. An associated fall in median Hib antibody concentrations occurred, from 1.29 μg/ml (95% confidence interval 0.90 to 1.64) in 1991 to 0.70 μg/ml (0.57 to 0.89) in 1994 (P = 0.006), with no significant change observed thereafter.Conclusions Although immunisation of infants resulted in an initial decline in Hib infections in adults, a resurgence in reported cases occurred in 2002-3. This rise was associated with an increase in cases in children and evidence of reduced immunity in older unimmunised cohorts. Childhood immunisation programmes may have unanticipated effects on the epidemiology of disease in older age groups, and surveillance strategies must be targeted at entire populations.     

Optimization of the lowry method of protein precipitation from theH. influenzae type b conjugate vaccine using deoxycholic acid and hydrochloric acid

The Lowry method was used in this study to measure protein inHaemophilus influenzae type b (Hib) conjugate vaccines (polyribosylibitol phosphate-tetanus toxoid; PRP-TT) using deoxycholic acid (DOC) to induce protein precipitation. Trichloroacetic acid (TCA) did not induce precipitation adequately from the Hib conjugate bulk and the freeze-dried Hib conjugate product. Its yield was approximately 50%. The matrix structure of Hib conjugate inhibits precipitation by TCA. Although the Lowry method can be carried out without precipitation in Hib conjugate bulk when no residual impurities (adipic acid dihydrazide [ADH], 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide-HCI [EDAC], phenol and cyanogens bromide [CNBr],etc.) are present, it cannot be used for Hib conjugate products that contain sucrose 8.5%, because 8.5% concentration of sucrose enhanced the protein concentration. DOC- and HCI-induced precipitation is an alternative method for evaluating the protein content of the Hib conjugate bulk and the Hib conjugate product. The precipitation was optimal with a final concentrate of 0.1% for DOC at 4°C and pH 2. This Lowry method, using DOC/HCI precipitation to induce protein precipitation, was confirmed a consistent, reproducible, and valid test for proteins in Hib conjugate bulk and its freeze-dried product.     

A hierarchical Bayesian model to predict the duration of immunity to Haemophilus influenzae type b

A hierarchical Bayesian regression model is fitted to longitudinal data on Haemophilus influenzae type b (Hib) serum antibodies. To estimate the decline rate of the antibody concentration, the model accommodates the possibility of unobserved subclinical infections with Hib bacteria that cause increasing concentrations during the study period. The computations rely on Markov chain Monte Carlo simulation of the joint posterior distribution of the model parameters. The model is used to predict the duration of immunity to subclinical Hib infection and to a serious invasive Hib disease.     

b型流感嗜血杆菌结合疫苗中载体蛋白质的效力研究

考察b型流感嗜血杆菌(Hib)结合疫苗的载体蛋白质—破伤风类毒素(TT)的免疫原性,为百白破与Hib四联疫苗中TT的使用提供参考。方法:将小鼠随机分为四组,分别注射Hib结合疫苗、Hib与百白混合疫苗、Hib与百白破混合疫苗、破伤风类毒素,比较它们在NIH小鼠体内所诱导产生的特异性抗体水平,并对这四组疫苗进行破伤风效力保护试验。结果表明,破伤风类毒素组,Hib与百白破混合疫苗组刺激的TT抗体水平远大于Hib结合疫苗组和Hib与百白混合疫苗组。效力试验虽四组间没有差异,只说明一定的抗体量就可以对小鼠提供保护。认为Hib结合疫苗中的载体蛋白并不能替代百白破疫苗中的破伤风类毒素。     

Diagnostic value of different laboratory methods in the diagnosis of pneumonia caused by Haemophilus influenzae type B

Comparative analysis of the diagnostic value of different laboratory methods in the diagnosis of H. influenzae b (Hib) pneumonia in children (bacteriological method, latex agglutination, counter immunoelectrophoresis, the passive hemagglutination test and the enzyme immunoassay (EIA) was carried out. EIA proved to be the most informative method for the diagnosing Hib pneumonia. EIA makes it possible to detect specific Hib antigens in different clinical materials in 48.8% of cases, as well as high titers of antibodies to mis infective agent in 61.7% of cases. The authors propose the unified criteria of the laboratory diagnosis of Hib infection in children.     

Process development of a New Haemophilus influenzae type b conjugate vaccine and the use of mathematical modeling to identify process optimization possibilities

Vaccination is one of the most successful public health interventions being a cost‐effective tool in preventing deaths among young children. The earliest vaccines were developed following empirical methods, creating vaccines by trial and error. New process development tools, for example mathematical modeling, as well as new regulatory initiatives requiring better understanding of both the product and the process are being applied to well‐characterized biopharmaceuticals (for example recombinant proteins). The vaccine industry is still running behind in comparison to these industries. A production process for a new Haemophilus influenzae type b (Hib) conjugate vaccine, including related quality control (QC) tests, was developed and transferred to a number of emerging vaccine manufacturers. This contributed to a sustainable global supply of affordable Hib conjugate vaccines, as illustrated by the market launch of the first Hib vaccine based on this technology in 2007 and concomitant price reduction of Hib vaccines. This paper describes the development approach followed for this Hib conjugate vaccine as well as the mathematical modeling tool applied recently in order to indicate options for further improvements of the initial Hib process. The strategy followed during the process development of this Hib conjugate vaccine was a targeted and integrated approach based on prior knowledge and experience with similar products using multi‐disciplinary expertise. Mathematical modeling was used to develop a predictive model for the initial Hib process (the ‘baseline’ model) as well as an ‘optimized’ model, by proposing a number of process changes which could lead to further reduction in price. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:568–580, 2016     

IGH V3-23*01 and its allele V3-23*03 differ in their capacity to form the canonical human antibody combining site specific for the capsular polysaccharide of Haemophilus influenzae type b

The IGH V3-23*01 gene is used in the formation of the canonical combining site which dominates the human antibody repertoire to the Haemophilus influenzae type b (Hib) polysaccharide (PS). An allele of the human IGH V3-23*01 gene, known as V3-23*03, differs from V3-23*01 in nine bases, eight of which are located in the second complementarity determining region. These eight differences encode five amino acid substitutions. In this study we investigated whether the V3-23*03 sequence polymorphism affected Hib PS binding. We constructed two Fab fragments that had the canonical Hib PS combining site VH-VL configuration but that had either V3-23*01 or V3-23*03. Radioantigen binding assay showed that on a concentration basis the V3-23*03 Fab was 20-fold more effective in binding Hib PS than the V3-23*01 Fab. The V3-23*03 Fab was 4-fold more effective than the V3-23*01 Fab in mediating facilitated bactericidal activity against Hib organisms. These findings identify a functional consequence of V3-23 allelism, and suggest that utilization of the V3-23*03 gene in the human Hib PS repertoire would generate canonical antibodies with higher affinity and protective efficacy than canonical antibodies utilizing V3-23*01. Thus, IGH V gene allelic variation has the potential to impact the generation of protective immunity to Hib.     

The 100 kDa haem:haemopexin-binding protein of Haemophilus Influenzae: structure and localization

All Haemophilus influenzae strains have an absolute requirement for exogenously supplied haem for aerobic growth. A majority of strains of H. influenzae type b (Hib) produce a 100 kDa protein which binds haem: haemopexin complexes. This 100 kDa haem:haemopexin binding protein, designated HxuA, was originally detected on the Hib cell surface. Monoclonal antibody (mAb)-based analyses revealed that the HxuA protein was also present in soluble form in Hib culture supernatants. This soluble HxuA protein exhibited haem:haemopexin-binding activity in a direct binding assay. Nucleotide sequence analysis of the hxuA gene from Hib strain DL42, together with N-terminal amino acid analysis of HxuA protein purified from Hib culture supernatant, revealed that this protein was synthesized as a 101 kDa precursor with a leader peptide that was removed to yield a 99kDa protein. Southern blot analysis of chromosomal DNA from four Hib and four non-typeable H. influenzae (NTHI) strains detected the presence of a single band in each strain that hybridized a Hib hxuA gene probe. Subsequent analysis of these NTHI strains showed that all four strains released into culture supernatant a haem:haemopexin-binding protein that migrated in SDS-PAGE at a rate similar or identical to that of the Hib HxuA protein. A Hib hxuA mutant was used to screen an NTHI genomic DNA library and an NTHI gene was cloned that complemented the mutation in this Hib strain. Nucleotide sequence analysis of this NTHI gene revealed that it encoded a protein with 87% identity to the Hib HxuA protein. The expression of HxuA by both Hib and NTHI strains indicates that this particular haem acquisition system is conserved among H. influenzae strains.     

Analysis of Haemophilus influenzae type b lipooligosaccharide-synthesis genes that assemble or expose a 2-keto-3-deoxyoctulosonic acid epitope

We recently isolated a recombinant phage from a Haemophilus influenzae type b (Hib) library that assembles an oligosaccharide with an apparent molecular weight of 1400 (1.4 K) on a 4.1 K Escherichia coli lipopolysaccharide (LPS) structure, producing a 5.5 K LPS species that contains a KDO (2-keto-deoxyoctulosonic acid) epitope. Subcloning and deletional analysis of the 14 kb Haemophilus insert showed that three overlapping restriction fragments contained within a 7.2 kb Pstl-BamHl fragment sequentially modified an E. coli 4.1 K LPS structure, generating novel species of 4.5 K, 5.1 K and 5.5 K. Only the 5.5 K species contained the KDO epitope. We confirmed the relationship between the cloned genes and Hib lipooligosaccharide (LOS) biosynthesis by constructing a mutant that expressed an altered LOS. Thus, the Hib 7.2 kb Pstl-BamHl restriction fragment contained a cluster of at least three genetic loci whose products acted sequentially in LOS biosynthesis.     

Haemophilus influenzae type b-outer membrane protein complex glycoconjugate vaccine induces cytokine production by engaging human toll-like receptor 2 (TLR2) and requires the presence of TLR2 for optimal immunogenicity

Conjugate vaccines consisting of the capsular polysaccharide (PS) of Haemophilus influenzae type b (Hib) covalently linked to carrier proteins, unlike pure PS, are immunogenic in infants and have significantly reduced Hib infections in the United States, but require multiple doses to induce protective anti-PS Ab titers. Hib-meningococcal outer membrane protein complex (OMPC) conjugate vaccine, however, elicits protective anti-PS Ab titers after one dose. We found that OMPC and Hib-OMPC engaged human Toll-like receptor 2 (TLR2) expressed in human embryonic kidney (HEK) cells, inducing IL-8 production, and engaged mouse TLR2 on bone marrow-derived dendritic cells, inducing TNF release. Hib conjugated to the carrier proteins CRM(197) and tetanus toxoid did not engage TLR2 on HEK or dendritic cells. Engagement of TLR2 by Hib-OMPC was MyD88 dependent, as Hib-OMPC-induced TNF production was ablated in MyD88 knockout (KO) mice. Hib-OMPC was significantly less immunogenic in TLR2 KO mice, inducing lower Hib PS IgG and IgM titers compared with those in wild-type mice. Splenocytes from OMPC-immunized TLR2 KO mice also produced significantly less IL-6 and TNF-alpha than those from wild-type mice. Hib-OMPC is unique among glycoconjugate vaccines by engaging TLR2, and the ability of Hib-OMPC to elicit protective levels of Abs after one dose may be related to TLR2-mediated induction and regulation of cytokines produced by T cells and macrophages in addition to the peptide/MHC II-dependent recruitment of T cell help commonly afforded by carrier proteins. TLR2 engagement by an adjuvant or carrier protein may be a useful strategy for augmentation of the anti-PS Ab response induced by glycoconjugate vaccines.     

Specific characteristics of composition of synthetic nutrient media for cultivation of Hemophilus influenzae type b from the standpoint of bacterial metabolism

In many countries vaccination against Haemophilus influenzae of type b (Hib) has permitted the liquidation of severe generalized forms of infections caused by these bacteria. The vaccine is obtained on the basis of Hib capsular polysaccharide. To obtain pure capsular polysaccharide, Hib should be cultivated on synthetic nutrient media. The present review deals with the data substantiating the advantages of using synthetic nutrient media for the cultivation of these bacteria with a view to obtaining pure capsular polysaccharide.     

Molecular cloning of a gene involved in lipooligosaccharide biosynthesis and virulence expression by Haemophilus influenzae type B

A wild-type Haemophilus influenzae type b (Hib) genomic DNA library was constructed in the plasmid shuttle vector pGJB103. A virulence-deficient lipooligosaccharide (LOS) mutant of Hib was used as a recipient for genetic transformation to screen this Hib genomic DNA library for genes involved in LOS expression. A recombinant plasmid containing a 7.8 kb PstI fragment of Hib DNA was shown to transform this LOS mutant to reactivity with a monoclonal antibody (mAb) specific for a wild-type LOS epitope. Transformation of two different virulence-deficient LOS mutants with a 4.4 kb BglII fragment of this recombinant plasmid yielded transformants which expressed LOS that bound the wild-type LOS-specific mAb and yielded profiles in sodium dodecyl sulphate/polyacrylamide gradient gel electrophoresis different from those of the original LOS mutants. These transformants with structurally altered LOS molecules also exhibited increased virulence in an animal model for invasive Hib disease. The virulence-transforming ability was further localized to a 1.8 kb BglII-AlwNI fragment of the Hib DNA insert. Nucleotide sequence analysis indicated the presence of a single large open reading frame within this fragment. This open reading frame contained 19 consecutive repeats of the tetramer CAAT near the 5' end. Linker insertion mutagenesis was used to demonstrate directly the involvement of this open reading frame in both LOS biosynthesis and virulence expression by Hib.     

Identification of two minor subunits in the pilus of Haemophilus influenzae.

Haemophilus influenzae type b (Hib) organisms produce pili, which mediate attachment to human cells and are multimeric structures composed of a 24-kDa subunit called pilin or HifA. Although pili from other organisms contain additional proteins accessory to pilin, no structural components other than pilin have been identified in Hib pili. Previous analysis of a Hib pilus gene cluster, however, suggested that two genes, hifD and hifE, may encode additional pilus subunits. To determine if hifD and hifE encode pilus components, the genes were overexpressed in Escherichia coli and the resulting proteins were purified and used to raise polyclonal antisera. Antisera raised against C-terminal HifD and HifE fragments reacted with H. influenzae HifD and HifE proteins, respectively, on Western immunoblots. Western immunoblot analysis of immunoprecipitated Hib pili demonstrated that HifD and HifE copurified with pili. In enzyme-linked immunosorbent assays, antisera raised against a recombinant HifE protein that contained most of the mature protein reacted more to piliated Hib than to nonpiliated Hib or to a mutant containing a hifE gene insertion. Immunoelectron microscopy confirmed that the HifE antiserum bound to pili and demonstrated that the antiserum bound predominantly to the pilus tips. These data indicate that HifD and HifE are pilus subunits. Adherence inhibition studies demonstrated that the HifE antiserum completely blocked pilus-mediated hemagglutination, suggesting that HifE mediates pilus adherence.     

Charged Residues in Surface-located Loops Influence Voltage Gating of Porin from Haemophilus influenzae Type b

Porin of Haemophilus influenzae type b (341 amino acids; M _r 37782) determines the permeability of the outer membrane to low molecular mass compounds. Purified Hib porin was subjected to chemical modification of lysine residues by succinic anhydride. Electrospray ionization mass spectrometry identified up to 12 modifications per porin molecule. Tryptic digestion of modified Hib porin followed by reverse phase chromatography and matrix assisted laser desorption ionization time-of-flight mass spectrometry mapped the succinylation sites. Most modified lysines are positioned in surface-located loops, numbers 1 and 4 to 7. Succinylated porin was reconstituted into planar lipid bilayers, and biophysical properties were analyzed and compared to Hib porin: there was an increased average single channel conductance compared to Hib porin (1.24+/−0.41 vs. 0.85+/−0.40 nanosiemens). The voltage-gating activity of succinylated porin differed considerably from that of Hib porin. The threshold voltage for gating was decreased from 75 to 40 mV. At 80 mV, steady-state conductance for succinylated porin was 50–55% of the instantaneous conductance. Hib porin at 80 mV showed a decrease to 89–91% of the instantaneous current levels. We propose that surface-located lysine residues are determinants of voltage gating for porin of Haemophilus influenzae type b. Received: 11 August 2000/Revised: 8 September 2000