Polysaccharide-protein Conjugate Vaccines Research Articles

Sensitive quantitation of low level free polysaccharide in conjugate vaccines by size exclusion chromatography-reverse phase liquid chromatography with UV detection

The level of free polysaccharide is a critical quality attribute of polysaccharide-protein conjugate vaccines. The work presented describes a simple and sensitive method for the determination of low level free polysaccharides in multiple polysaccharide-protein conjugates. The method utilizes a reverse phase (RP) column to perform a size exclusion chromatography (SEC) separation of free polysaccharide and a reverse phase liquid chromatography (RPLC) separation of free protein and protein-polysaccharide conjugate. The use of phosphate buffer in the mobile phase enables the universal and sensitive detection of low level free polysaccharides at UV 200 nm. The method has been validated to monitor low level free polysaccharide (<1 %) in multiple polysaccharide-protein conjugates. The limit of quantitation is 2 μg/ml or 0.3 % free polysaccharide in 0.6 mg/ml polysaccharide-protein conjugate. The accuracy is in the range of 94.1.0–108.5 %.

Persistence of Pneumococcal Antibodies After Primary Immunisation With a Polysaccharide–Protein Conjugate Vaccine

P Zimmerman, KP Perrett, G Berbers, N Curtis. Arch Dis Child. 2019;104(7):680–684 The pneumococcal polysaccharide-protein conjugate vaccine (PCV) is a part of the primary vaccination series recommended by the World Health Organization (WHO) as well as the Centers for Disease Control and Prevention (CDC). The purpose of this study was to examine the protective antibody response at 7 and 13 months to PCV by following the primary vaccination series recommended by the WHO, using the 3 + 0 schedule (PCV at 6 weeks, 3–4 months, and 4–6 months of age). Patients were gathered from a previous patient population that was a part of the Melbourne Infant Study: BCG for Allergy and Infection Reduction that was looking at BCG immunization at birth and the protective nature against allergy, childhood infection, and asthma from whom blood samples were collected at 7 and/or 13 months of age. All of these infants were in Melbourne, Australia, and were a part of the Australian National Immunization program from 2013 to 2016. Inclusion criteria were infants born at >32 weeks’ gestation, birth weight >1500 g, mother who was …

Developing a serocorrelate of protection against invasive group B streptococcus disease in pregnant women: a feasibility study.

Group B streptococcus is the leading cause of infection in infants. Currently, intrapartum antibiotic prophylaxis is the major strategy to prevent invasive group B streptococcus disease. However, intrapartum antibiotic prophylaxis does not prevent maternal sepsis, premature births, stillbirths or late-onset disease. Maternal vaccination may offer an alternative strategy. Multivalent polysaccharide protein conjugate vaccine development is under way and a serocorrelate of protection is needed to expedite vaccine licensure. The ultimate aim of this work is to determine the correlate of protection against the major group B streptococcus disease-causing serotypes in infants in the UK. The aim of this feasibility study is to test key operational aspects of the study design. Prospective cohort study of pregnant women and their infants in a 6-month period (1 July to 31 December 2018). Five secondary and tertiary hospitals from London and South England. National iGBS disease surveillance was conducted in all trusts in England and Wales. Pregnant women aged ≥ 18 years who were delivering at one of the selected hospitals and who provided consent during the study period. There were no exclusion criteria. No interventions were performed. (1) To test the feasibility of collecting serum at delivery from a large cohort of pregnant women. (2) To test the key operational aspects for a proposed large serocorrelates study. (3) To test the feasibility of collecting samples from those with invasive group B streptococcus. A total of 1823 women were recruited during the study period. Overall, 85% of serum samples were collected at three sites collecting only cord blood. At the two sites collecting maternal, cord and infant blood samples, the collection rate was 60%. A total of 614 women were screened for group B streptococcus with a colonisation rate of 22% (serotype distribution: 30% III, 25% Ia, 16% II, 14% Ib, 14% V and 1% IV). A blood sample was collected from 34 infants who were born to colonised women. Maternal and infant blood and the bacterial isolates for 15 newborns who developed invasive group B streptococcal disease during the study period were collected (serotype distribution: 29% III, 29% II, 21% Ia, 7% Ib, 7% IV and 7% V). Recruitment and sample collection were dependent on the presence of research midwives rather than the whole clinical team. In addition, individualised consent limited the number of women who could be approached each day, and site set-up for the national surveillance study and the limited time period of this feasibility study limited recruitment of all eligible participants. We have verified the feasibility of collecting and processing rectovaginal swabs and blood samples in pregnant women, as well as samples from those with invasive group B streptococcal disease. We have made recommendations for the recruitment of cases within the proposed GBS3 study and for controls both within GBS3 and as an extension of this feasibility study. A large case-control study comparing specific immunoglobulin G levels in mothers whose infants develop invasive group B streptococcal disease with those in colonised mothers whose infants do not develop invasive group B streptococcal disease is recommended. Current Controlled Trials ISRCTN49326091; IRAS project identification number 246149/REC reference number 18/WM/0147. This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 23, No. 67. See the NIHR Journals Library website for further project information.

Development and pre-clinical evaluation of a synthetic oligosaccharide-protein conjugate vaccine against Neisseria meningitidis serogroup C

Significant improvement has been made in the development of vaccines against Neisseria meningitidis infections since the introduction of polysaccharide-protein conjugate vaccines. Conventional bacterial capsular polysaccharide (PS) based conjugate vaccines require unique and expensive manufacturing facilities, complex production processes and extensive quality testing. Synthetic oligosaccharide (OS) based approach is one of the novel technologies that is being developed to simplify production of conjugate vaccines. OSs can be chemically synthesized to a desired length long enough to represent the antigenic epitopes which often present as a homogenous mixture. We prepared OSs corresponding to tetramer and octamer of N. meningitidis serogroup C (MenC) PS by organic synthesis. The MenC tetramer and octamer were further conjugated with tetanus toxoid to produce respective monovalent conjugates having the desired physico-chemical characteristics. The conjugates were evaluated in a mouse model for immunogenicity and compared with a licensed PS conjugate vaccine. Synthetic conjugates could induce anti-MenC PS IgG as well as serum bactericidal titers at levels comparable to those elicited by the licensed vaccine. The increase in length of synthetic oligomers from tetramer to octamer did not appear to increase immunogenicity. The results establish the pre-clinical proof of concept for a synthetic MenC oligosaccharide conjugate vaccine candidate.

Effect on meningococcal serogroup W immunogenicity when Tdap was administered prior, concurrent or subsequent to the quadrivalent (ACWY) meningococcal CRM197-conjugate vaccine in adult Hajj pilgrims: A randomised controlled trial

Immune responses to the capsular polysaccharide administered in the polysaccharide-protein conjugate vaccines can be either improved or suppressed by the pre-existence of immunity to the carrier protein. Receiving multiple vaccinations is essential for travellers such as Hajj pilgrims, and the use of conjugated vaccines is recommended.We studied the immune response to meningococcal serogroup W upon prior, concurrent and sequential administration of a quadrivalent meningococcal conjugate vaccine (MCV4) conjugated to CRM197 (coadministered with 13 valent pneumococcal vaccine conjugate CRM197 [PCV13]), and tetanus-diphtheria-acellular pertussis (Tdap) vaccine in Australian adults before attending the Hajj pilgrimage in 2014.Participants were randomly assigned, by computer-generated numbers, to three study arms by 1:1:1 ratio. Group A received Tdap followed by MCV4-CRM197 (+PCV13) 3–4 weeks later. Group B received all three vaccines in a single visit. Group C received MCV4-CRM197 (+PCV13) followed by Tdap 3–4 weeks later. Blood samples obtained prior to and 3–4 weeks after immunisation with MCV4-CRM197 were tested for meningococcal serogroup W-specific serum bactericidal antibody responses using baby rabbit complement (rSBA).One hundred and seven participants aged between 18 and 64 (median 40) years completed the study. No significant difference in meningococcal serogroup W rSBA geometric mean titre (GMT) was observed between the study arms post vaccination with MCV-CRM197 but Group A tended to have a slightly lower GMT (A = 404, B = 984 and C = 1235, p = 0.15). No statistical difference was noticed between the groups in proportions of subjects achieving a ≥4-fold rise in rSBA titres or achieving rSBA titre ≥8 post vaccination.In conclusion, receipt of MCV4-CRM197 vaccine prior, concurrent or subsequent to Tdap has similar immunologic response, and hence concurrent administration is both immunogenic and practical. However, further investigation into whether carrier induced suppression is a public health issue is suggested.Clinical trial registration: ANZCTR no. ACTRN12613000536763.

The Association Between Breast Milk Group B Streptococcal Capsular Antibody Levels and Late-onset Disease in Young Infants.

Animal-model studies have demonstrated less group B streptococcal (GBS) invasive disease and gastrointestinal colonization after enteral administration of serotype-specific capsular antibodies. There is, however, a paucity of information on the association of breast milk GBS serotype-specific capsular antibodies and risks for invasive disease in infants. The aim of this study was to explore the association between natural secretory immunoglobulin A (sIgA) capsular antibodies in breast milk and the occurrence of late-onset disease (LOD) in young infants. A matched case-control study was undertaken in infants <3 months of age in Johannesburg, South Africa. Breast milk samples were collected on cases and controls matched for gestational age, maternal age, and human immunodeficiency virus status at time of enrollment. Capsular serotype Ia, Ib, III, and V sIgA antibody concentrations were measured using the fluorescence-based micro-bead immunosorbent assay. Breast milk samples were available for 31 LOD cases (8 serotype Ia and 23 serotype III), 21 recto-vaginally colonized matched controls (10 serotype Ia and 11 serotype III), and 84 serotype Ia and 105 serotype III noncolonized matched controls. Using a Bayesian model to estimate the probability of disease, there were 90% reductions in the risks of developing serotypes Ia and III LOD with sIgA concentrations ≥0.14 µg/mL and ≥2.52 µg/mL, respectively. Breast milk sIgA capsular antibodies were associated with lower risks for LOD in young infants. The ability of GBS polysaccharide-protein conjugate vaccines currently under development to induce sIgA responses warrant investigation as potential mediators of protection against LOD.

Impact of HIV-1 infection and antigen class on T follicular helper (TFH) cell responses to pneumococcal polysaccharide-protein conjugate vaccine (PCV-13)

Abstract Pneumococcal infections are common and serious complications of HIV-1 disease. Prevention has been compromised by the limited magnitude and quality of antibody responses to T cell-independent (TI-2) pneumococcal capsular polysaccharides (PPS). The PCV-13 vaccine conjugates PPS to a T cell-dependent (TD) protein (diphtheria toxoid; DT). We investigated the differential response to PPS and DT by antibody-secreting B cells (ASC) after immunization with PCV-13 in newly-diagnosed healthy HIV+ and control adults. The numbers of PPS-specific IgG ASC increased significantly and similarly in HIV+ and controls 1 week after vaccination. However, DT-specific IgG ASC increased significantly in controls but not HIV+ subjects. To determine the cellular basis of these disparate responses to DT and PPS, we characterized the frequency and activation of T follicular helper (TFH) cells, the predominant T cell subset providing B cell help. The percentages of and intracellular IL-21 in TFH cells were unchanged with vaccination. However, expression of ICOS, which sustains TFH function and phenotype, increased significantly after vaccination among controls, but not the HIV+ group. Increases in ICOS+ TFH correlated with changes in TD DT-specific IgG ASC in controls but not in HIV+. In contrast, ICOS expression did not correlate with TI-2 PPS-specific ASC in either group. Levels of TFH differentiating factors IL-12 and IL-6 increased post vaccination only in controls. In summary, although TI-2 PPS-specific responses were similar in both groups and independent of TFH activation, TFH ICOS-expression was most closely related to responses to TD DT, which were compromised even in recently diagnosed healthy HIV+ adults.

OC 8465 THE DEVELOPMENT OF A CONJUGATE VACCINE AGAINST GROUP B STREPTOCOCCUS: AN AFRICAN PERSPECTIVE

BackgroundStreptococcus agalactiae, or group B streptococcus (GBS), is a gram-positive streptococcal bacterium that is well-recognised as one of the leading causes of infant death, particularly in the early neonatal period (the first week of life). An estimated one in five pregnant women around the world carries GBS bacteria in their gastrointestinal or genitourinary tracts and vertical transmission from colonised mothers can lead to invasive disease in their offspring. A recent study conservatively estimated that out of 410,000 GBS cases globally every year, there are at least 1 47 000 stillbirths and infant deaths. Despite being home to only 13% of the world’s population, Africa has the highest GBS disease burden, with 54% of estimated cases and 65% of stillbirths and infant deaths.An effective GBS vaccine, given during pregnancy, is a promising strategy to protect against GBS disease. Currently, no licensed vaccine exists to prevent it, but scientific evaluation of feasibility is favourable. The leading vaccine candidates are capsular polysaccharide-protein conjugate vaccines. Evidence suggests maternal immunisation with a safe and effective GBS vaccine may reduce the disease risk in neonates and young infants.The Biovac Institute was established as a private-public partnership and is the only Southern African vaccine manufacturer. Located in Cape Town, South Africa, Biovac’s mission is to become a leading vaccine developer and producer in South Africa to increase capacity in Africa which only has four other vaccine manufacturers.In collaboration with PATH, an international health organisation, and other partners, Biovac is developing a multivalent conjugate vaccine against GBS. The first stage of the project involves the development of biopharmaceutical manufacturing processes and analytical tests, the preparation of clinical trial product, and execution of a first-in-human clinical trial.This presentation will provide an overview of the project, progress to date, and the path to commercialisation.

Persistence of pneumococcal antibodies after primary immunisation with a polysaccharide–protein conjugate vaccine

IntroductionDespite immunisation, antibiotics and intensive care management, infection with Streptococcus pneumoniae remains a major cause of morbidity and mortality in children. The WHO currently recommends vaccinating infants with either a...

A multicenter clinical study on 1 138 cases of invasive pneumococcal disease in children from 2012 to 2017

Objective: To explore the clinical features, the serotype distribution and drug resistance of the isolates in patient with invasive pneumococcal disease (IPD). Methods: By retrieving the laboratory information system in 18 children's hospitals from 2012 to 2017, the children with IPD were enrolled. Streptococcus pneumoniae (Spn) must be isolated from the sterile sites (blood, cerebrospinal fluid, hydrothorax and joint effusion etc.). The clinical characteristics, serotype, drug resistance, treatment and prognosis were reviewed and analyzed. According to the telephone follow up results, the patients were divided into death group and recovered group. The index as an independent risk factor of mortality was demonstrated by multivariate logistic regression analysis. Results: There were 1 138 children with IPD, including 684 male and 454 female. The proportion of male to female was 1.5∶1. The age ranged from one day to 16 years. The median age was 1 year 3 month. The majority was under 5 years of age (89.3%, n= 1 016), especially under 2 years of age (61.9%, n=704). In all cases, 88.2% (n=1 004) were community acquired infection. The infections included meningitis (n=446, 39.2%), pneumonia with bacteremia (n=339, 29.8%), and bacteremia without focus (n=232, 20.4%). Underlying diseases were found in 242 cases (21.3%). Co-infections were determined in 62 cases (5.4%) with mycoplasma, 27 cases (2.4%) with adenovirus and 34 cases with influenza virus (3.0%). The penicillin insensitivity (PNSP) rates in meningitis and non-meningitis isolates were 69.5% (276/397) and 35.9% (221/615), respectively. There were 81 strains serotyped, in which 93.8% (76/81) were covered by 13-valent protein-polysaccharide conjugate vaccine (PCV13). In the 965 patients who were followed up by phone call, 156 cases (16.2%) were confirmed dead. The independent risk factors for the death were under 2 years of age (OR=2.143, 95%CI 1.284-3.577, P=0.004), meningitis (OR=3.066, 95%CI 1.852-5.074, P<0.01), underlying disease (OR=4.801, 95%CI 2.953-7.804, P<0.01), septic shock(OR=3.542, 95%CI 1.829-6.859, P<0.01), disseminated intravascular coagulation (DIC) (OR=4.150, 95%CI 1.468-11.733, P=0.007), multiple organ failure (OR=12.693, 95%CI 6.623-24.325, P<0.01) and complications of central nervous system (OR=1.975, 95%CI 1.144-3.410, P=0.015). Conclusions: Most children with IPD were under 5 years of age, having underlying diseases and acquired the infection in community. The independent risk factors for death were under two years old, meningitis, underlying diseases and multiple organ failure. The problem of drug resistance was severe. The universal immunization of PCV13 would be effective to prevent IPD in Chinese children.

Protection Against Invasive Infections in Children Caused by Encapsulated Bacteria.

The encapsulated bacteria Streptococcus pneumoniae, Neisseria meningitis, Haemophilus influenzae, and Streptococcus agalactiae (Group B Streptococcus) have been responsible for the majority of severe infections in children for decades, specifically bacteremia and meningitis. Isolates which cause invasive disease are usually surrounded by a polysaccharide capsule, which is a major virulence factor and the key antigen in protective protein-polysaccharide conjugate vaccines. Protection against these bacteria is largely mediated via polysaccharide-specific antibody and complement, although the contribution of these and other components, and the precise mechanisms, vary between species and include opsonophagocytosis and complement-dependent bacteriolysis. Further studies are required to more precisely elucidate mechanisms of protection against non-type b H. influenzae and Group B Streptococcus.

Nasal immunization with recombinant chimeric pneumococcal protein and cell wall from immunobiotic bacteria improve resistance of infant mice to Streptococcus pneumoniae infection.

Respiratory tract infections and invasive disease caused by Streptococcus pneumoniae in high-risk groups are a major global health problem. Available human vaccines have reduced immunogenicity and low immunological memory in these populations, as well as high cost as a public health strategy in poor communities. In addition, no single pneumococcal protein antigen has been able to elicit protection comparable to that achieved using protein-polysaccharide conjugate vaccines. In this context, chimeric pneumococcal proteins raise as potential good vaccine candidates because of their simplicity of production and reduced cost. The aim of this work was to study whether the nasal immunization of infant mice with the recombinant chimeric pneumococcal protein (PSFP) was able to improve resistance to S. pneumoniae, and whether the immunomodulatory strain Lactobacillus rhamnosus CRL1505 or its cell wall (CW1505) could be used as effective mucosal adjuvants. Our results showed that the nasal immunization with PSPF improved pneumococcal-specific IgA and IgG levels in broncho-alveolar lavage (BAL), reduced lung bacterial counts, and avoided dissemination of pneumococci into the blood. Of interest, immunization with PSPF elicited cross-protective immunity against different pneumococcal serotypes. It was also observed that the nasal immunization of infant mice with PSPF+CW1505 significantly increased the production of pneumococcal-specific IgA and IgG in BAL, as well as IgM and IgG in serum when compared with PSPF alone. PSPF+CW1505 immunization also improved the reduction of pneumococcal lung colonization and its dissemination in to the bloodstream when compared to PSPF alone. Our results suggest that immunization with PSPF together with the cell wall of the immunomodulatory strain L. rhamnosus CRL1505 as a mucosal adjuvant could be an interesting alternative to improve protection against pneumococcal infection in children.

Group B Streptococcus (GBS) vaccine

Intrapartum antibiotic prophylaxis (IPA) during labor for women identified as having recto-vaginal Groups B Streptococcus (GBS) colonization, has been successful in preventing early-onset invasive (<7 days of age) disease in new-borns in high income countries where implemented. Nevertheless, GBS remains a leading cause of neonatal sepsis in high-income settings, and is now also the leading cause of bacterial meningitis in USA. Also, the incidence of invasive GBS disease remains high in sub-Saharan African countries, where IAP is not feasible, whilst its contribution to neonatal sepsis is uncertain in South Asia. Furthermore, recent data indicate that the burden of GBS associated stillbirths might be similar to that for EOD in some African countries. Recent advances in the prevention of invasive GBS disease during early infancy, including the potential to prevent late onset disease (7-89 days age) which is not preventable by IAP, is the development of polyvalent polysaccharide protein conjugate vaccines. Recent studies have reported on the safety and immunogenicity of a trivalent (serotypes Ia, Ib and III) GBS polysaccharide protein conjugate vaccine (GBS-3CV). This vaccine elicits immune responses in pregnant women vaccinated during the last trimester, albeit less so among women in whom the pre-vaccination antibody titers were less than the lower detection limit of the assay. The ratio of antibody transfer to the newborns’ of these women was approximately 0.5-0.7, indicating less efficient transplacental transfer of polysaccharide targeted IgG than is observed for IgG targeted at protein antigens. Furthermore, vaccination of HIV-infected women, elicited lower quantitative antibody responses than in HIV-uninfected women, suggesting the need for different vaccine schedules in these women. Also, the GBS conjugate vaccine appears to be more immunogenic in African than European/North American women. The licensure of a GBS vaccine for pregnant women aimed at protection against invasive GBS disease of their newborns will, however, require sample size in excess of 70,000 participants for an invasive disease endpoint. An alternate licensure pathway, as is the case for meningococcal vaccine, could be premised on establishing a sero-correlate of protection against invasive disease and using this to license the vaccine based on immunogenicity and safety.

Effect of Tdap upon antibody response to meningococcal polysaccharide when administered before, with or after the quadrivalent meningococcal TT-conjugate vaccine (coadministered with the 13-valent pneumococcal CRM197-conjugate vaccine) in adult Hajj pilgrims: A randomised controlled trial

Hajj pilgrims are susceptible to several serious infections and are required to receive multiple vaccinations. Polysaccharide-protein conjugate vaccines contain carrier proteins such as tetanus toxoid (TT), diphtheria toxoid or a mutant of diphtheria toxoid (CRM197). These carrier proteins may interact with other conjugate or combination vaccines containing tetanus or diphtheria on concurrent or sequential administration. We examined the immune interaction of separate and concomitant administration of a tetanus/diphtheria/acellular pertussis (Tdap) vaccine with a TT-conjugated quadrivalent meningococcal vaccine (MCV4) (coadministered with 13-valent pneumococcal CRM197-conjugate vaccine [PCV13]) in adult Australian pilgrims before attending Hajj in 2015.We randomly assigned each participant to one of three vaccination schedules. Group 1 received Tdap 3–4 weeks before receiving MCV4 coadministered with PCV13. Group 2 received all three vaccines concomitantly. Group 3 received MCV4 and PCV13 3–4 weeks before Tdap. Blood samples were collected at baseline, at each vaccination visit and 3–4 weeks after vaccination and tested for response to meningococcal serogroups C, W and Y using a serum bactericidal antibody (rSBA) assay with baby rabbit complement, and to diphtheria and tetanus toxoid, measuring IgG antibodies by ELISA. Participants completed symptom diaries after each vaccination. A total of 166 participants aged 18–64 (median 42) years were recruited, of whom 160 completed the study. Compared to the other groups, Group 1 (given Tdap first) had significantly lower proportion of subjects achieving a ≥4-fold rise in rSBA for serogroup W. No difference was detected across the three groups in achieving protection threshold (rSBA ≥8 post vaccination) or SBA geometric mean titre (GMT) post vaccination.Group 3, which was given MCV4/PCV13 first, had high levels of antibody against diphtheria and tetanus than the other groups, when tested prior to receipt of Tdap; Only the anti-tetanus responses remained significantly higher after Tdap administration. No serious adverse events were reported.In conclusion, when multiple vaccination is required for Hajj pilgrims, administering Tdap concurrently with MCV4/PCV13 produces adequate immune responses, and avoids meningococcal immune interference, in the convenience of a single consultation. However, giving Tdap 3–4 weeks after MCV4/PCV13 has the advantage of an enhanced tetanus toxoid response.The trial is Trials Registry (ANZCTR): ACTRN12613000536763.

Production and efficacy of a low-cost recombinant pneumococcal protein polysaccharide conjugate vaccine

Streptococcus pneumoniae is the leading cause of bacterial pneumonia. Although this is a vaccine preventable disease, S. pneumoniae still causes over 1 million deaths per year, mainly in children under the age of five. The biggest disease burden is in the developing world, which is mainly due to unavailability of vaccines due to their high costs. Protein polysaccharide conjugate vaccines are given routinely in the developed world to children to induce a protective antibody response against S. pneumoniae. One of these vaccines is Prevnar13, which targets 13 of the 95 known capsular types. Current vaccine production requires growth of large amounts of the 13 serotypes, and isolation of the capsular polysaccharide that is then chemically coupled to a protein, such as the diphtheria toxoid CRM197, in a multistep expensive procedure. In this study, we design, purify and produce novel recombinant pneumococcal protein polysaccharide conjugate vaccines in Escherichia coli, which act as mini factories for the low-cost production of conjugate vaccines. Recombinant vaccine efficacy was tested in a murine model of pneumococcal pneumonia; ability to protect against invasive disease was compared to that of Prevnar13. This study provides the first proof of principle that protein polysaccharide conjugate vaccines produced in E. coli can be used to prevent pneumococcal infection. Vaccines produced in this manner may provide a low-cost alternative to the current vaccine production methodology.

Analytical Comparability Assessments of 5 Recombinant CRM197 Proteins From Different Manufacturers and Expression Systems

Cross-reacting material 197 (CRM197), a single amino acid mutant of diphtheria toxoid, is a commonly used carrier protein in commercial polysaccharide protein conjugate vaccines. In this study, CRM197 proteins from 3 different expression systems and 5 different manufacturers were obtained for an analytical comparability assessment using a wide variety of physicochemical and in vitro antigenic binding assays. A comprehensive analysis of the 5 CRM197 molecules demonstrate that recombinant CRM197's expressed in heterologous systems (Escherichia coli and Pseudomonas fluorescens) are overall highly similar (if not better in some cases) to those expressed in the traditional system (Corynebacterium diphtheriae) in terms of primary sequence/post-translational modifications, higher order structural integrity, apparent solubility, physical stability profile (vs. pH and temperature), and in vitro antigenicity. These results are an encouraging step to demonstrate that recombinant CRM197 expressed in alternative sources have the potential to replace CRM197 expressed in C diphtheriae as a source of immunogenic carrier protein for lower cost polysaccharide conjugate vaccines. The physicochemical assays established in this work to monitor the key structural attributes of CRM197 should also prove useful as complementary characterization methods (to routine quality control assays) to support future process and formulation development of lower cost CRM197 carrier proteins for use in various conjugate vaccines.

Changes in the Nature and Severity of Invasive Pneumococcal Disease in Children Before and After the Seven-valent and Thirteen-valent Pneumococcal Conjugate Vaccine Programs in Calgary, Canada.

Since the introduction of childhood pneumococcal conjugate vaccines, invasive pneumococcal disease (IPD) incidence has decreased in children and the predominant serotypes causing disease have changed. This study describes changes in the clinical features of IPD in children (<18 years) before and after the conjugate vaccine introduction. The Calgary Area Streptococcus pneumoniae Epidemiology Research study collects information on all IPD cases in Calgary, Alberta, Canada. Descriptive and regression analyses were used to compare IPD in the pre-vaccine (January 2000 to August 2002), post-7-valent protein-polysaccharide conjugate vaccine (September 2002 to June 2010) and post-13-valent protein-polysaccharide conjugate vaccine (PCV13) (July 2010 to December 2015) periods; intensive care unit and inpatient admissions were outcome measures. The incidence of IPD in children (<18 years) decreased from an average of 17 cases/100,000/yr in 2000-2001 to 4 cases/100,000/yr in 2015. The median age of children presenting with IPD shifted from 2.0 years (interquartile range: 2.5) in the pre-vaccine period to 3.9 years (interquartile range: 6.2) in the post-PCV13 period. The proportion of children with a comorbidity that is an indication for pneumococcal vaccination did not change. Invasive disease with focus (meningitis, pneumonia, empyema, peritonitis) compared with invasive disease with bacteremia only increased from 44.6% in pre-vaccine to 64.0% and 61.4% in the post-7-valent protein-polysaccharide conjugate vaccine and post-PCV13 periods, respectively (P = 0.017). Having IPD in the post-PCV13 period compared with the pre-vaccine period was associated with an increased odds of hospitalization [Odds ratio (OR): 2.9; 95% Confidence Interval (CI): 1.4-6.2]. Clinical features of IPD have changed since pneumococcal conjugate vaccines were introduced, with a shift toward more focal infections requiring hospitalization. Although overall IPD cases have declined, disease that does occur appears to be more severe.

Brucellosis: Improved Diagnostics and Vaccine Insights from Synthetic Glycans.

ConspectusBrucellosis is a serious zoonotic bacterial disease that is ranked by the World Health Organization among the top seven “neglected zoonoses” that threaten human health and cause poverty. It is a costly, highly contagious disease that affects ruminants, cattle, sheep, goats, and other productive animals such as pigs. Symptoms include abortions, infertility, decreased milk production, weight loss, and lameness. Brucellosis is also the most common bacterial disease that is transmitted from animals to humans, with approximately 500 000 new human cases each year. Detection and slaughter of infected animals is required to eradicate the disease, as vaccination alone is currently insufficient. However, as the most protective vaccines compromise serodiagnosis, this creates policy dilemmas, and these often result in the failure of eradication and control programs. Detection of antibodies to the Brucella bacterial cell wall O-polysaccharide (OPS) component of smooth lipopolysaccharide is used in diagnosis of this disease, and the same molecule contributes important protective efficacy to currently deployed veterinary whole-cell vaccines. This has set up a long-standing paradox that while Brucella OPS confers protective efficacy to vaccines, its presence results in similar antibody profiles in infected and vaccinated animals. Consequently, differentiation of infected from vaccinated animals (DIVA) is not possible, and this limits efforts to combat the disease. Recent clarification of the chemical structure of Brucella OPS as a block copolymer of two oligosaccharide sequences has provided an opportunity to utilize unique oligosaccharides only available via chemical synthesis in serodiagnostic tests for the disease. These oligosaccharides show excellent sensitivity and specificity compared with the native polymer used in current commercial tests and have the added advantage of assisting discrimination between brucellosis and infections caused by several bacteria with OPS that share some structural features with those of Brucella. During synthesis and immunochemical evaluation of these synthetic antigens, it became apparent that an opportunity existed to create a polysaccharide–protein conjugate vaccine that would not create antibodies that give false positive results in diagnostic tests for infection. This objective was reduced to practice, and immunization of mice showed that antibodies to the Brucella A antigen could be developed without reacting in a diagnostic test based on the M antigen. A conjugate vaccine of this type could readily be developed for use in humans and animals. However, as chemical methods advance and modern methods of bacterial engineering mature, it is expected that the principles elucidated by these studies could be applied to the development of an inexpensive and cost-effective vaccine to combat endemic brucellosis in animals.

Effect of Vaccine-Elicited Antibodies on Colonization of Neisseria meningitidis Serogroup B and C Strains in a Human Bronchial Epithelial Cell Culture Model

ABSTRACTCapsular polysaccharide-protein conjugate vaccines protect individuals from invasive disease and decrease carriage, which reduces spread of the organism in the population. In contrast, antibodies elicited by plain polysaccharide or protein antigen-based meningococcal (Men) vaccines have little or no effect on decreasing carriage. In this study, we investigated the mechanism by which vaccine-induced human immunoglobulin G (IgG) antibodies affect colonization by meningococcal serogroup B (MenB) or C (MenC) strains using a human bronchial epithelial cell culture model (16HBE14o-). Fluorescence microscopy showed that bacteria colonizing the apical side of 16HBE14o- monolayers had decreased capsular polysaccharide on the bacterial surface that resulted from shedding the capsule and not decreased production of polysaccharide. Capsular polysaccharide shedding depended on the presence of 16HBE14o- cells and bacteria but not direct adherence of the bacteria to the cells. Treatment of bacteria and cells with postimmunization MenC-conjugate IgG or murine anti-MenB polysaccharide monoclonal antibodies (MAbs) inhibited capsule shedding, microcolony dispersal, and invasion of the 16HBE14o- cell monolayer. In contrast, the IgG responses elicited by immunization with MenC polysaccharide (PS), MenB outer membrane vesicle (OMV)-based, or factor H binding protein (FHbp)-based vaccines were not different than preimmune IgG or no-treatment response. The results provide new insights on the mechanism by which high-avidity anticapsular antibodies elicited by polysaccharide-conjugate vaccines affect meningococcal colonization. The data also suggest that any effect on colonization by IgG elicited by OMV- or FHbp-based vaccines may involve a different mechanism.

Defective anti-polysaccharide IgG vaccine responses in IgA deficient mice.

We report that IgA-/- mice exhibit specific defects in IgG antibody responses to various polysaccharide vaccines (Francisella tularensis LPS and Pneumovax), but not protein vaccines such as Fluzone. This defect further included responses to polysaccharide-protein conjugate vaccines (Prevnar and Haemophilus influenzae type b-tetanus toxoid vaccine). In agreement with these findings, IgA-/- mice were protected from pathogen challenge with protein- but not polysaccharide-based vaccines. Interestingly, after immunization with live bacteria, IgA+/+ and IgA-/- mice were both resistant to lethal challenge and their IgG anti-polysaccharide antibody responses were comparable. Immunization with live bacteria, but not purified polysaccharide, induced production of serum B cell-activating factor (BAFF), a cytokine important for IgG class switching; supplementing IgA-/- cell cultures with BAFF enhanced in vitro polyclonal IgG production. Taken together, these findings show that IgA deficiency impairs IgG class switching following vaccination with polysaccharide antigens and that live bacterial immunization can overcome this defect. Since IgA deficient patients also often show defects in antibody responses following immunization with polysaccharide vaccines, our findings could have relevance to the clinical management of this population.