Interlaboratory comparison of a multiplex immunoassay that measures human serum IgG antibodies against six-group B streptococcus polysaccharides.

Measurement of IgG antibodies against group B streptococcus (GBS) capsular polysaccharide (CPS) by use of a standardized and internationally accepted multiplex immunoassay is important for the evaluation of candidate maternal GBS vaccines in order to compare results across studies. A standardized assay is also required if serocorrelates of protection against invasive GBS disease are to be established in infant sera for the six predominant GBS serotypes since it would permit the comparison of results across the six serotypes. We undertook an interlaboratory study across five laboratories that used standardized assay reagents and protocols with a panel of 44 human sera to measure IgG antibodies against GBS CPS serotypes Ia, Ib, II, III, IV, and V. The within-laboratory intermediate precision, which included factors like the lot of coated beads, laboratory analyst, and day, was generally below 20% relative standard deviation (RSD) for all six serotypes, across all five laboratories. The cross-laboratory reproducibility was < 25% RSD for all six serotypes, which demonstrated the consistency of results across the different laboratories. Additionally, anti-CPS IgG concentrations for the 44-member human serum panel were established. The results of this study showed assay robustness and that the resultant anti-CPS IgG concentrations were reproducible across laboratories for the six GBS CPS serotypes when the standardized assay was used.

Mucosal Immunization Has Benefits over Traditional Subcutaneous Immunization with Group A Streptococcus Antigens in a Pilot Study in a Mouse Model.

Group A Streptococcus (GAS) is a major human pathogen for which there is no licensed vaccine. To protect against infection, a strong systemic and mucosal immune response is likely to be necessary to prevent initial colonization and any events that might lead to invasive disease. A broad immune response will be necessary to target the varied GAS serotypes and disease presentations. To this end, we designed a representative panel of recombinant proteins to cover the stages of GAS infection and investigated whether mucosal and systemic immunity could be stimulated by these protein antigens. We immunized mice sublingually, intranasally and subcutaneously, then measured IgG and IgA antibody levels and functional activity through in vitro assays. Our results show that both sublingual and intranasal immunization in the presence of adjuvant induced both systemic IgG and mucosal IgA. Meanwhile, subcutaneous immunization generated only a serum IgG response. The antibodies mediated binding and killing of GAS cells and blocked binding of GAS to HaCaT cells, particularly following intranasal and subcutaneous immunizations. Further, antigen-specific assays revealed that immune sera inhibited cleavage of IL-8 by SpyCEP and IgG by Mac/IdeS. These results demonstrate that mucosal immunization can induce effective systemic and mucosal antibody responses. This finding warrants further investigation and optimization of humoral and cellular responses as a viable alternative to subcutaneous immunization for urgently needed GAS vaccines.

Progress towards a glycoconjugate vaccine against Group A Streptococcus.

The Group A Carbohydrate (GAC) is a defining feature of Group A Streptococcus (Strep A) or Streptococcus pyogenes. It is a conserved and simple polysaccharide, comprising a rhamnose backbone and GlcNAc side chains, further decorated with glycerol phosphate on approximately 40% GlcNAc residues. Its conservation, surface exposure and antigenicity have made it an interesting focus on Strep A vaccine design. Glycoconjugates containing this conserved carbohydrate should be a key approach towards the successful mission to build a universal Strep A vaccine candidate. In this review, a brief introduction to GAC, the main carbohydrate component of Strep A bacteria, and a variety of published carrier proteins and conjugation technologies are discussed. Components and technologies should be chosen carefully for building affordable Strep A vaccine candidates, particularly for low- and middle-income countries (LMICs). Towards this, novel technologies are discussed, such as the prospective use of bioconjugation with PglB for rhamnose polymer conjugation and generalised modules for membrane antigens (GMMA), particularly as low-cost solutions to vaccine production. Rational design of "double-hit" conjugates encompassing species specific glycan and protein components would be beneficial and production of a conserved vaccine to target Strep A colonisation without invoking an autoimmune response would be ideal.

Sublingual immunisation with GBS serotype III capsular polysaccharide-tetanus toxoid conjugate vaccine induces systemic and mucosal antibody responses which are opsonophagocytic and inhibit GBS colonisation of vaginal epithelial cells.

No vaccines are currently licensed against Group B streptococcus (GBS), an important cause of morbidity and mortality in babies and adults. Using a mouse model, and in vitro opsonophagocytosis and colonisation assays, we evaluated the potential of a sublingually-administered polysaccharide-conjugate vaccine against GBS serotype III. Sublingual immunisation of mice with 10 µg of GBS conjugate vaccine once a week for 5 weeks induced a substantial systemic IgG anti-polysaccharide response which was similar to the level induced by subcutaneous immunsation. In addition, sublingual immunisation also induced mucosal (IgA) antibody responses in the mouth, intestines and vagina. Immune sera and intestinal washes were functionally active at mediating killing of the homologous GBS serotype III in an opsonophagocytosis assay. In addition, intestinal and vaginal washes inhibited the colonisation of mouse vaginal epithelial cells by the vaccine homologous strain. These results suggest that, in addition to the induction of high levels of IgG antibodies that could be transduced from the immunised mother to the foetus to protect the newborn against GBS infection, sublingual immunisation can elicit a substantial mucosal antibody response which might play an important role in the prevention of GBS colonisation in immunised women, thereby eliminating the risk of GBS transmission from the mother to the baby during pregnancy or at birth.

GMMA as an Alternative Carrier for a Glycoconjugate Vaccine against Group A Streptococcus.

Group A Streptococcus (GAS) causes about 500,000 annual deaths globally, and no vaccines are currently available. The Group A Carbohydrate (GAC), conserved across all GAS serotypes, conjugated to an appropriate carrier protein, represents a promising vaccine candidate. Here, we explored the possibility to use Generalized Modules for Membrane Antigens (GMMA) as an alternative carrier system for GAC, exploiting their intrinsic adjuvant properties. Immunogenicity of GAC-GMMA conjugate was evaluated in different animal species in comparison to GAC-CRM197; and the two conjugates were also compared from a techno-economic point of view. GMMA proved to be a good alternative carrier for GAC, resulting in a higher immune response compared to CRM197 in different mice strains, as verified by ELISA and FACS analyses. Differently from CRM197, GMMA induced significant levels of anti-GAC IgG titers in mice also in the absence of Alhydrogel. In rabbits, a difference in the immune response could not be appreciated; however, antibodies from GAC-GMMA-immunized animals showed higher affinity toward purified GAC antigen compared to those elicited by GAC-CRM197. In addition, the GAC-GMMA production process proved to be more cost-effective, making this conjugate particularly attractive for low- and middle-income countries, where this pathogen has a huge burden.

Multicomponent Vaccines against Group A Streptococcus Can Effectively Target Broad Disease Presentations.

Group A Streptococcus (GAS) is an important global human pathogen, with a wide range of disease presentations, from mild mucosal infections like pharyngitis to invasive diseases such as toxic shock syndrome. The effect on health and mortality from GAS infections is substantial worldwide, particularly from autoimmune sequelae-like rheumatic heart disease (RHD), and there is currently no licenced vaccine. We investigated protein antigens targeting a broad range of GAS disease presentations as vaccine components in individual and combination formulations. The potency and functional immunity generated were evaluated and compared between groups. Antibodies against all components were found in pooled human IgG (IVIG) and an immune response generated following the subcutaneous immunisation of mice. A combination immunisation showed a reduction in IgG response for SpyCEP but an increase for Cpa and Mac-1 (IdeS). An opsonophagocytosis assay (OPA) showed the killing of GAS with immune sera against M protein and combination groups, with a lower killing activity observed for immune sera against other individual antigens. Specific antigen assays showed functional immunity against SpyCEP and Mac-1 from both individual and combination immunisations, with the activity correlating with antibody titres. However, efficient blocking of the binding activity of Cpa to collagen I and fibronectin could not be demonstrated with immune sera or purified IgG. Our data indicate that combination immunisations, while effective at covering a broader range of virulence factors, can also affect the immune response generated. Further, our results showed that an OPA alone is inadequate for understanding protection from vaccination, particularly when considering protection from immune evasion factors and evaluation of the colonisation leading to pharyngitis.

Impact of routine vaccination against Haemophilus influenzae type b in The Gambia: 20 years after its introduction.

BACKGROUND: In 1997, The Gambia introduced three primary doses of Haemophilus influenzae type b (Hib) conjugate vaccine without a booster in its infant immunisation programme along with establishment of a population-based surveillance on Hib meningitis in the West Coast Region (WCR). This surveillance was stopped in 2002 with reported elimination of Hib disease. This was re-established in 2008 but stopped again in 2010. We aimed to re-establish the surveillance in WCR and to continue surveillance in Basse Health and Demographic Surveillance System (BHDSS) in the east of the country to assess any shifts in the epidemiology of Hib disease in The Gambia. METHODS: In WCR, population-based surveillance for Hib meningitis was re-established in children aged under-10 years from 24 December 2014 to 31 March 2017, using conventional microbiology and Real Time Polymerase Chain Reaction (RT-PCR). In BHDSS, population-based surveillance for Hib disease was conducted in children aged 2-59 months from 12 May 2008 to 31 December 2017 using conventional microbiology only. Hib carriage survey was carried out in pre-school and school children from July 2015 to November 2016. RESULTS: In WCR, five Hib meningitis cases were detected using conventional microbiology while another 14 were detected by RT-PCR. Of the 19 cases, two (11%) were too young to be protected by vaccination while seven (37%) were unvaccinated. Using conventional microbiology, the incidence of Hib meningitis per 100 000-child-year (CY) in children aged 1-59 months was 0.7 in 2015 (95% confidence interval (CI) = 0.0-3.7) and 2.7 (95% CI = 0.7-7.0) in 2016. In BHDSS, 25 Hib cases were reported. Nine (36%) were too young to be protected by vaccination and five (20%) were under-vaccinated for age. Disease incidence peaked in 2012-2013 at 15 per 100 000 CY and fell to 5-8 per 100 000 CY over the subsequent four years. The prevalence of Hib carriage was 0.12% in WCR and 0.38% in BHDSS. CONCLUSIONS: After 20 years of using three primary doses of Hib vaccine without a booster Hib transmission continues in The Gambia, albeit at low rates. Improved coverage and timeliness of vaccination are of high priority for Hib disease in settings like Gambia, and there are currently no clear indications of a need for a booster dose.

Chitosan nanoparticle antigen uptake in epithelial monolayers can predict mucosal but not systemic in vivo immune response by oral delivery.

This study compared in vitro and in vivo antigen delivery effects of ultrapure chitosan (CS) chloride. CS nanoparticles were formulated to incorporate ovalbumin (OVA) as a model antigen and characterised for size, charge, OVA complexation and release. The effect of CS:OVA nanoparticles on cell viability, epithelial tight junctions and transepithelial permeation of OVA was tested on Caco-2 monolayer in vitro intestinal model. The system's ability to elicit immune responses was subsequently tested in vivo. The work confirmed that CS complexes with OVA into nano-size entities. Nanocomplexes displayed favourable delivery properties, namely OVA release and no notable cytotoxicity. CS:OVA markedly enhanced antigen delivery across Caco-2 monolayers. However, the system did not elicit notable in vivo immune responses (some mucosal response was apparent) following oral delivery. The study highlights that a clear effect on antigen permeability across epithelial monolayers in vitro may predict the in vivo mucosal but not systemic immune response following oral delivery.

Nano-sized Soluplus® polymeric micelles enhance the induction of tetanus toxin neutralising antibody response following transcutaneous immunisation with tetanus toxoid.

The use of Soluplus® polymeric micelles as a novel adjuvant for tetanus toxoid (TTxd) in transcutaneous immunisation was evaluated. TTxd was added to Soluplus® polymeric micelles to form TTxd-Soluplus® nano-aggregates with a size of 68nm. Non-adjuvanted TTxd commonly induces very poor antibody response by the transcutaneous route. However, in this study, the use of TTxd-Soluplus® resulted in a significant increase in the antibody response to TTxd, which was similar to that induced in the presence of CPG-oligodeoxynucleotides (CPG-ODNs) adjuvant. The toxin neutralising potency of the immune sera induced by TTxd-Soluplus® was also much stronger than that from TTxd alone, in a passive transfer experiment in mice. Soluplus® also enhanced the immunogenicity of the toxoid when TTxd-Soluplus® was stored at 4°C for 4weeks, but not at higher temperatures. Confocal microscopy imaging showed a much higher uptake of TTxd in the epidermis and dermis layers of the skin when it was associated with Soluplus®, suggesting that the mechanism for Soluplus® adjuvanticity is through enhanced uptake of the TTxd through the skin. Overall, our findings demonstrated that Soluplus® is an effective novel adjuvant for transcutaneous immunisation.

Mucosal Antibodies to the C Terminus of Toxin A Prevent Colonization of Clostridium difficile.

Mucosal immunity is considered important for protection against Clostridium difficile infection (CDI). We show that in hamsters immunized with Bacillus subtilis spores expressing a carboxy-terminal segment (TcdA26-39) of C. difficile toxin A, no colonization occurs in protected animals when challenged with C. difficile strain 630. In contrast, animals immunized with toxoids showed no protection and remained fully colonized. Along with neutralizing toxins, antibodies to TcdA26-39 (but not to toxoids), whether raised to the recombinant protein or to TcdA26-39 expressed on the B. subtilis spore surface, cross-react with a number of seemingly unrelated proteins expressed on the vegetative cell surface or spore coat of C. difficile These include two dehydrogenases, AdhE1 and LdhA, as well as the CdeC protein that is present on the spore. Anti-TcdA26-39 mucosal antibodies obtained following immunization with recombinant B. subtilis spores were able to reduce the adhesion of C. difficile to mucus-producing intestinal cells. This cross-reaction is intriguing yet important since it illustrates the importance of mucosal immunity for complete protection against CDI.

International collaborative study for establishment of the 2nd WHO International Standard for Haemophilus influenzae type b polysaccharide.

In this report we present the results of a collaborative study for the preparation and calibration of a replacement International Standard (IS) for Haemophilus influenzae type b polysaccharide (polyribosyl ribitol phosphate; 5-d-ribitol-(1 â†’ 1)-ß-d-ribose-3-phosphate; PRP). Two candidate preparations were evaluated. Thirteen laboratories from 9 different countries participated in the collaborative study to assess the suitability and determine the PRP content of two candidate standards. On the basis of the results from this study, Candidate 2 (NIBSC code 12/306) has been established as the 2nd WHO IS for PRP by the Expert Committee of Biological Standards of the World Health Organisation with a content of 4.904 ± 0.185mg/ampoule, as determined by the ribose assays carried out by 11 of the participating laboratories.

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.

Immunogenicity and physico-chemical characterisation of a candidate conjugate vaccine against group B streptococcus serotypes Ia, Ib and III.

The immunogenicity and physico-chemical characteristics of a candidate conjugate vaccine against group B streptococcus serotypes Ia, Ib and III, were evaluated. The level and functional activity of serotype-specific antibody responses induced by monovalent and combined formulations were investigated using a mouse model and in vitro opsonophagocytosis assay. Molecular sizing of the conjugates and integrity of the intermediate components were evaluated by optical spectroscopy and size exclusion chromatography. All three serotypes induced substantial antibody responses which were functionally active. Combining the three serotypes did not seem to affect the antibody responses to individual serotypes, except when given at high dose, where the IgG response to serotype III but not the opsonophagocytic activity was slightly reduced compared to monovalent administration.

Immunogenicity and thermal stability of a combined vaccine against Haemophilus influenzae type b and Neisseria meningitidis serogroup C diseases.

The immunogenicity, structure and stability of a combined conjugate vaccine against Haemophilus influenzae type b and meningococcal serogroup C (Hib/MenC) were investigated. A rat model for immunogenicity showed that antibody responses to Hib and MenC in the combined vaccine were similar to or higher than those of individual conjugates given alone, or concomitantly at separate sites. At elevated temperatures, the combination vaccine was slightly more stable than a monovalent Hib-TT vaccine, with respect to molecular size, which could be attributed to differences in the formulations. Following 5 weeks incubation at 56 degrees C, there was some dissociation of high molecular weight conjugate without significant loss of saccharide integrity; however, this did not significantly affect the vaccine immunogenicity, demonstrating the stability of this lyophilized vaccine.

Current progress with Moraxella catarrhalis antigens as vaccine candidates.

The success of the immunization programs against Haemophilus influenzae type b and, more recently, Streptococcus pneumoniae in developed and some developing countries has demonstrated that invasive disease caused by these bacteria can be very effectively controlled by vaccination. There is also evidence that pneumococcal vaccines can reduce the incidence of acute otitis media in children. More complete control of this disease would be achieved if infections caused by Moraxella catarrhalis and nontypeable H. influenzae, the other common agents of otitis media in children and of a number of respiratory-associated infections in both children and adults, could also be controlled. Since these bacteria do not possess capsules and are not known to secrete exotoxins, the search for vaccine candidates has focused on the conserved epitopes exposed on the bacterial outer membrane. In this article, we review the contribution of M. catarrhalis to disease and recent advances in the development and testing of various vaccine candidates against this bacterium, including those still in the development stage and those approaching clinical trials. Recommendations are proposed for approaches needed for the standardization of assays and use of appropriate animal models for quality-control testing of these vaccine candidates. Regulatory issues surrounding vaccines of this type are also discussed.

Transcutaneous delivery of tetanus toxin Hc fragment induces superior tetanus toxin neutralizing antibody response compared to tetanus toxoid.

Transcutaneous immunization is a promising vaccination delivery strategy which targets potent immune cells residing in the outer layer of the skin. In this study, the immunogenicity and neutralizing potency of the non-toxic Hc fragment of tetanus toxin (HcWT) and a mutant of Hc lacking ganglioside binding activity were compared with that of tetanus toxoid (TTxd) following transcutaneous immunization (TCI) of mice. Mice immunized with HcWT in the absence of an adjuvant induced highest anti-toxoid and anti-Hc antibody titres, with a significant increase in the toxin neutralizing antibody response compared with TTxd. These results are in contrast to previous studies employing subcutaneous delivery, where TTxd was found to be a more potent immunogen than the Hc fragment of the toxin. We conclude that the HcWT protein is more immunogenic than TTxd when given via the transcutaneous route. Our results suggest that TCI may provide an opportunity for effective delivery of toxin-like antigens which harbor protective epitopes and that traditional toxoid proteins may not be optimal antigens for skin immunization.

A retrospective study on the quality of Haemophilus influenzae type b vaccines used in the UK between 1996 and 2004.

Following the reduction in efficacy of Hib-TT vaccines in the primary immunization schedule observed in the UK between 1999 and 2003, batches of vaccine manufactured by two different companies were retrospectively examined by the National Institute for Biological Standards and Control. The study evaluated 41 batches of the Hib-TT vaccines manufactured between 1994 and 2003, assaying potency (total PRP saccharide content), integrity (% free saccharide), consistency (molecular sizing), and immunogenicity, as well as reviewing data previously obtained at the time of release. The study indicated the stability of the lyophilized final fill vaccines to extend well past their assigned shelf-lives, and found no trends in the endotoxin content, total saccharide or % free saccharide content. A trend towards slightly larger conjugates was observed over time in Hib-TT A, evidenced in both the manufacturer's data obtained at the time that samples were submitted for testing and in data obtained from the retrospective analysis. The study confirmed that that there had been no significant change in the quality of the Hib vaccines that could possibly account for the change reported in their protective efficacy in the UK. The study also demonstrated the value of independent testing of vaccines from the time of licensure and in the ongoing monitoring and re-examination of selected batches, as necessary, to assure their continuing quality, safety and consistency.

Physico-chemical characterisation and immunogenicity of a multi-valent candidate vaccine against non-typeable Haemophilus influenzae and Moraxella catarrhalis.

The physico-chemical characteristics and immunogenicity of a candidate vaccine against otitis media, prepared from recombinant lipidated outer membrane proteins (rLP4 and rLP6) from non-typeable Haemophilus influenzae (NTHi) and of the ubiquitous cell surface protein UspA2 from Moraxella catarrhalis, were evaluated. Optical spectroscopy, size exclusion chromatography and gel electrophoresis were used to characterise the purified protein components and assess their purity and molecular sizes. The results showed that the three proteins were highly purified. Possible dimers in rLP4, dimers and multimers in rLP6 and UspA2 were detected. Small amounts of rLP4 and rLP6 dimers and most of UspA2 complexes remained tightly bound even after SDS treatment under reducing conditions. Immunogenicity studies showed that all proteins induced substantial antibody responses in mice immunised with AlPO4-adsorbed rLP4, rLP6 or UspA2 or a combination of these proteins. However, combination of these proteins resulted in a reduced response to rLP4 and rLP6, but not to UspA2, suggesting interference between these proteins which should be taken into consideration during the development and evaluation of this vaccine.

Evaluation of the saccharide content and stability of the first WHO International Standard for Haemophilus influenzae b capsular polysaccharide.

Haemophilus influenzae b conjugate vaccines (Hib) are almost entirely evaluated by physico-chemical methods to ensure the consistency of manufacture of batches. As different assays are employed for the quantification of Hib capsular polysaccharide PRP (polyribosyl ribitol phosphate; 5-D-ribitol-(1-->1)-beta-D-ribose-3-phosphate) in final formulations and bulk components, there was deemed a need for an International Standard of Hib PRP polysaccharide to be made available. Ten laboratories from 8 different countries participated in a collaborative study to determine the PRP content and assess the suitability of a candidate International Standard PRP preparation (02/208). The results illustrate that a reduction in between-laboratory variability could be achieved by use of a common reference preparation and data analysis showed no significant differences in the values obtained by the different assays: ribose, phosphorus, and high performance anion exchange chromatography-pulsed amperometric detection (HPAEC-PAD), suggesting the suitability of the proposed reference for use across these assays for quantification of PRP content in Hib vaccines. On the basis of the results of this study, the First International Standard for PRP, NIBSC Code 02/208, has been established by the Expert Committee of Biological Standards of the World Health Organisation, with a content of 4.933+/-0.267mg/ampoule, as determined by the ribose assays carried out by 7 of the participating laboratories.

Immune interaction between components of acellular pertussis-diphtheria-tetanus (DTaP) vaccine and Haemophilus influenzae b (Hib) conjugate vaccine in a rat model.

We have previously shown that, consistent with clinical trial results, the immune response to a Haemophilus influenzae b (Hib) conjugate vaccine in a rat model was compromised and modulated when given combined with a DTaP3 vaccine, as compared to both vaccines given separately. The present study extended our investigation to evaluate the immunogenicity of all DTaP3 components in combined versus separate administration of Hib with DTaP3 and investigated immune interactions between Hib and individual components of DTaP3. Rats were immunised with Hib and DTaP3 or with Hib and individual DTaP3 components. Cellular and humoral immune responses to Hib and DTaP3 components were evaluated. Our results indicate that the immunogenicity of DTaP3 components was similar or greater in combined versus separate administration of Hib and DTaP3. Moreover, combined administration of Hib and TT reduced immunogenicity of both Hib and TT. Hib immunogenicity was also significantly reduced when given combined with FHA and following adsorption to Al(OH)3.