Capsular Polysaccharide Conjugate Vaccines Research Articles

Development and validation of a 6-plex Luminex-based assay for measuring human serum antibodies to group B streptococcus capsular polysaccharides

ABSTRACT Six serotypes (Ia, Ib, II, III, IV, and V) cause nearly all group B streptococcal (GBS) disease globally. Capsular polysaccharide (CPS) conjugate vaccines aim to prevent GBS disease, however, licensure of a vaccine would depend on a standardized serological assay for measuring anti-CPS IgG responses. A multiplex direct Luminex-based immunoassay (dLIA) has been developed to simultaneously measure the concentration of serum IgG specific for the six prevalent GBS CPS serotypes. Assay validation was performed using serum samples obtained from human subjects vaccinated with an investigational 6-valent GBS CPS conjugate vaccine. Results for the assay are expressed as IgG concentrations (µg/mL) using a human serum reference standard composed of pooled sera from vaccinated subjects. The lower limits of quantitation (LLOQ) for all serotypes covered in the 6-plex GBS IgG dLIA fell within the range of 0.002-0.022 µg/mL IgG. Taken together, the 6-plex GBS IgG dLIA platform is specific for the six GBS serotypes included in Pfizer’s investigational vaccine, has a wide dilution adjusted assay range, and is precise (<18.5% relative standard deviation) for all serotypes, and, therefore, is suitable for quantitatively measuring vaccine-induced or naturally acquired serotype-specific anti-CPS IgG responses against GBS.

Immune responses against group B Streptococcus monovalent and pentavalent capsular polysaccharide tetanus toxoid conjugate vaccines in Balb/c mice

Immunization of pregnant women with Group B Streptococcus (GBS) capsular polysaccharide (CPS) conjugate vaccine (CV) could protect young infants against invasive GBS disease. We evaluated the immunogenicity of investigational five GBS monovalent (serotypes Ia, Ib, II, III, and V) CPS-tetanus toxoid (TT)-CV with adjuvant and GBS pentavalent CPS-TT-CV with adjuvant (GBS5-CV-adj) and without adjuvant (GBS5-CV-no-adj), in Balb/c mice. Aluminum phosphate was the adjuvant in the formulations, where included. The homotypic immunoglobulin G (IgG) geometric mean concentration (GMC) and opsonophagocytic activity (OPA) geometric mean titer (GMT) did not differ after the third dose of the GBS5-CV-adj vaccine compared with the monovalent counterparts for all five serotypes. The GBS5-CV-adj induced higher post-vaccination serotype-specific IgG GMCs and OPA GMTs compared to GBS5-CV-no_adj. The GBS5-CV with and without adjuvant should be considered for further development as a potential vaccine for pregnant women to protect their infants against invasive GBS disease.

Vi polysaccharide and conjugated vaccines afford similar early, IgM or IgG-independent control of infection but boosting with conjugated Vi vaccines sustains the efficacy of immune responses.

Vaccination with Vi capsular polysaccharide (Vi-PS) or protein-Vi typhoid conjugate vaccine (TCV) can protect adults against Salmonella Typhi infections. TCVs offer better protection than Vi-PS in infants and may offer better protection in adults. Potential reasons for why TCV may be superior in adults are not fully understood. Here, we immunized wild-type (WT) mice and mice deficient in IgG or IgM with Vi-PS or TCVs (Vi conjugated to tetanus toxoid or CRM197) for up to seven months, with and without subsequent challenge with Vi-expressing Salmonella Typhimurium. Unexpectedly, IgM or IgG alone were similarly able to reduce bacterial burdens in tissues, and this was observed in response to conjugated or unconjugated Vi vaccines and was independent of antibody being of high affinity. Only in the longer-term after immunization (>5 months) were differences observed in tissue bacterial burdens of mice immunized with Vi-PS or TCV. These differences related to the maintenance of antibody responses at higher levels in mice boosted with TCV, with the rate of fall in IgG titres induced to Vi-PS being greater than for TCV. Therefore, Vi-specific IgM or IgG are independently capable of protecting from infection and any superior protection from vaccination with TCV in adults may relate to responses being able to persist better rather than from differences in the antibody isotypes induced. These findings suggest that enhancing our understanding of how responses to vaccines are maintained may inform on how to maximize protection afforded by conjugate vaccines against encapsulated pathogens such as S. Typhi.

Isolation and Characterization of Human Monoclonal Antibodies to Pneumococcal Capsular Polysaccharide 3.

ABSTRACTThe current pneumococcal capsular polysaccharide (PPS) conjugate vaccine (PCV13) is less effective against Streptococcus pneumoniae serotype 3 (ST3), which remains a major cause of pneumococcal disease and mortality. Therefore, dissecting structure-function relationships of human ST3 pneumococcal capsular polysaccharide (PPS3) antibodies may reveal characteristics of protective antibodies. Using flow cytometry, we isolated PPS3-binding memory B cells from pneumococcal vaccine recipients and generated seven PPS3-specific human monoclonal antibodies (humAbs). Five humAbs displayed ST3 opsonophagocytic activity, four induced ST3 agglutination in vitro, and four mediated both activities. Two humAbs, namely, C10 and C27, that used the same variable heavy (VH) and light (VL) chain domains (VH3-9*01/VL2-14*03) both altered ST3 gene expression in vitro; however, C10 had fewer VL somatic mutations, higher PPS3 affinity, and promoted in vitro ST3 opsonophagocytic and agglutinating activity, whereas C27 did not. In C57BL/6 mice, both humAbs reduced nasopharyngeal colonization with ST3 A66 and a clinical strain, B2, and prolonged survival following lethal A66 intraperitoneal infection, but only C10 protected against lethal intranasal infection with the clinical strain. After performing VL swaps, C10VH/C27VL exhibited reduced ST3 binding and agglutination, but C27VH/C10VL binding was unchanged. However, both humAbs lost the ability to reduce colonization in vivo when their light chains were replaced. Our findings associate the ability of PPS3-specific humAbs to reduce colonization with ST3 agglutination and opsonophagocytic activity, and reveal an unexpected role for the VL in their functional activity in vitro and in vivo. These findings also provide insights that may inform antibody-based therapy and identification of surrogates of vaccine efficacy against ST3.IMPORTANCE Despite the global success of vaccination with pneumococcal conjugate vaccines, serotype 3 (ST3) pneumococcus remains a leading cause of morbidity and mortality. In comparison to other vaccine-included serotypes, the ST3 pneumococcal capsular polysaccharide (PPS3) induces a weaker opsonophagocytic response, which is considered a correlate of vaccine efficacy. Previous studies of mouse PPS3 monoclonal antibodies identified ST3 agglutination as a correlate of reduced ST3 nasopharyngeal colonization in mice; however, neither the agglutinating ability of human vaccine-elicited PPS3 antibodies nor their ability to prevent experimental murine nasopharyngeal colonization has been studied. We generated and analyzed the functional and in vivo efficacy of human vaccine-elicited PPS3 monoclonal antibodies and found that ST3 agglutination associated with antibody affinity, protection in vivo, and limited somatic mutations in the light chain variable region. These findings provide new insights that may inform the development of antibody-based therapies and next-generation vaccines for ST3.

Distribution of Capsular Types of Campylobacter jejuni Isolates from Symptomatic and Asymptomatic Children in Peru.

.Campylobacter jejuni is the leading bacterial cause of diarrhea worldwide. A capsular polysaccharide (CPS) conjugate vaccine is under development and requires determination of the valency. However, distribution of CPS types circulating globally is presently poorly described. We aimed to determine whether CPS type distribution in Peru differs from that in other endemic regions. We used a multiplex polymerase chain reaction (PCR) assay for the detection of CPS encoding genes capable of distinguishing all 35 CPS types on Campylobacter isolates in two prospective communities based studies conducted in cohorts of children less than 59 months of age in Peru. Results showed that CPS type HS4 complex was the most prevalent, followed by HS3 complex and HS15. Differences in CPS type for symptomatology were not statistically significant. Most subjects demonstrated repeated infections over time with different CPS types, suggesting that CPS types may confer of a level of homologous protective immunity. In this dataset, some differences in CPS type distribution were observed in comparison to other low-middle income countries. Further studies need to be conducted in endemic areas to increase our knowledge of CPS type distribution and guide vaccine development.

A Novel Hexavalent Capsular Polysaccharide Conjugate Vaccine (GBS6) for the Prevention of Neonatal Group B Streptococcal Infections by Maternal Immunization.

BackgroundGroup B streptococcus (GBS) causes serious diseases in newborn infants, often resulting in lifelong neurologic impairments or death. Prophylactic vaccination of pregnant women prior to delivery could provide comprehensive protection, as early onset and late-onset disease and maternal complications potentially could be addressed.MethodsCapsular polysaccharide conjugate vaccine GBS6 was designed using surveillance data yielded by whole-genome sequencing of a global collection of recently recovered GBS isolates responsible for invasive neonatal GBS disease. Capsular polysaccharides were isolated, oxidized using sodium periodate, and conjugated to CRM197 by reductive amination in dimethyl sulfoxide. Immune responses in mice and rhesus macaques were measured in a multiplex Luminex immunoglobulin G (IgG) assay and opsonophagocytic activity assays.ResultsThe optimized conjugates were immunogenic, alone and in combination, in mice and rhesus macaques, inducing IgG antibodies that mediated opsonophagocytic killing. Active immunization of murine dams with GBS6 prior to mating resulted in serotype-specific protection of pups from a lethal challenge with GBS. Protection following passive administration of serotype-specific IgG monoclonal antibodies to dams demonstrated conclusively that anticapsular polysaccharide IgG alone is sufficient for protection.ConclusionsThe findings support the ongoing clinical evaluation of maternal GBS6 vaccination as a potential alternative method to prevent GBS disease in infants.

Evaluation of two WHO First International Standards for Vi polysaccharide from Citrobacter freundii and Salmonella enterica subspecies enterica serovar Typhi

Numerous Vi capsular polysaccharide (Vi PS) conjugate vaccines to protect young children and infants from Typhoid are either licensed or under development. These vaccines are evaluated by laboratory methods to ensure their potency and that quality requirement are met. International Standard (IS) preparations of Vi PS are needed to calibrate and harmonise these assays. Twenty laboratories from 12 countries participated in a collaborative study to evaluate two candidate ISs: Citrobacter freundii Vi PS (NIBSC code 12/244) and Salmonella enterica serovar Typhi Vi PS (16/126). On the basis of returned results and stability profiles, these standards were established by the WHO Expert Committee on Biological Standardization in Oct 2017 as the First WHO IS for C. freundii Vi PS with a content of 1.94 ± 0.12 mg Vi PS per ampoule (expanded uncertainty with coverage factor of k = 2.11 corresponding to a 95% level of confidence) and the First WHO IS for S. Typhi Vi PS with a content of 2.03 ± 0.10 mg Vi PS per ampoule (expanded uncertainty with coverage factor of k = 2.11), as determined by quantitative NMR. The study also showed the ISs are suitable for physicochemical and immuno assays used for the quantitation of the Vi PS component in Vi PS and conjugate vaccines.

Establishment of the first International Standard for human anti-typhoid capsular Vi polysaccharide IgG

Vi capsular polysaccharide (Vi) conjugate vaccines, which can prevent typhoid in infants and young children, are being developed. Comparative immunogenicity studies are facilitated by an International Standard (IS) for human anti-Vi IgG. 16/138, a pool of sera from volunteers which received either Vi conjugate vaccine or plain Vi vaccine, was assessed as an IS alongside U.S. reference reagent Vi-IgGR1, 2011. Samples were tested in a commercial ELISA (n = 7), a standardised ELISA based on biotinylated Vi (n = 7) and in-house ELISAs (n = 7). Valid estimates were obtained for the potency of all samples in the commercial ELISA, and the commutability of 16/138 and Vi-IgGR1, 2011 was evident for the commercial ELISA and in-house ELISAs based on a coating of Vi and protein. The WHO Expert Committee on Biological Standardization established 16/138 as the first IS for anti-Vi IgG with 100 IU per ampoule and assigned 163 IU per vial of Vi-IgGR1, 2011.

Design and Biological Assembly of Polyester Beads Displaying Pneumococcal Antigens as Particulate Vaccine

Streptococcus pneumoniae can cause life-threatening infections mostly in infants, children, and elderly people. Capsular polysaccharide conjugate vaccines provide serotype-dependent protection against S. pneumoniae infections but fail to protect against new emerging serotypes. To overcome these limitations, pneumolysin (Ply), a serotype-independent and conserved protein was selected. As such subunit vaccines lack immunogenicity, we engineered Ply to be attached to self-assembled polyester beads in order to boost immunogenicity. To display Ply at the surface of these polyester beads, it was translationally fused to the N-terminus of the polyhydroxybutyrate (PHB) synthase (PhaC), which mediates PHB bead assembly inside recombinant Escherichia coli. We also chemically conjugated the capsular polysaccharide (CPS) 19F to isolated PHB beads to further assess their antigen carrier properties. CPS conjugated to soluble tetanus toxoid served as control. Balb/c mice immunized with Ply-PhaC beads and 19F-PhaC beads induced specific and higher IgG levels than the respective soluble counterparts. The induced IgG antibodies recognized Ply in whole cell lysates of six different serotypes of S. pneumoniae. Additionally, restimulated splenocytes from animals immunized with Ply-PhaC beads produced a balanced INF-γ/IL-17A profile unlike animals immunized with soluble Ply. The 19F-PhaC beads induced production of antibodies showing high opsonophagocytic titers against the homologous strain, serotype 19F, while CPS 19F only mixed with PhaC beads did not elicit any detectable immune response. This study provided insight into the design of PHB beads as a carrier of proteinaceous antigens and CPS in order to induce immune responses for the prevention of pneumococcal infections.

From research to licensure and beyond: clinical development of MenB-FHbp, a broadly protective meningococcal B vaccine

ABSTRACTIntroduction: Given the characteristics of meningococcal carriage and transmission and the sudden, often severe onset and long-term consequences of disease, vaccination can most effectively provide large-scale control of invasive disease. Six serogroups (A, B, C, W, X, and Y) cause nearly all meningococcal disease globally. Capsular polysaccharide conjugate vaccines can prevent serogroups A, C, W, and Y disease. More recently, recombinant protein vaccines for preventing serogroup B meningococcal (MenB) disease have become available, with a major target of vaccine-induced immune response for both vaccines being bacterial factor H binding protein (FHbp). Importantly, FHbp segregates into only two distinct subfamilies (A [also classified as variants 2 and 3] and B [variant 1]). This review summarizes the complete clinical development program supporting licensure of MenB-FHbp (Trumenba®, Bivalent rLP2086), the only MenB vaccine containing antigens from both FHbp subfamilies.Areas covered: Eleven published clinical studies assessing MenB-FHbp efficacy and safety among 20,803 adolescents and adults are examined. Particular focus is on the methodology of immunogenicity assessments used as a surrogate for clinical efficacy.Expert commentary: Clinical studies in adolescents and adults consistently demonstrated MenB-FHbp safety and induction of immunologic responses against antigenically and epidemiologically diverse MenB isolates, supporting licensure and immunization recommendations.

WITHDRAWN: Establishment of the first International Standard for human anti-typhoid capsular Vi polysaccharide IgG

Abstract Vi capsular polysaccharide (Vi) conjugate vaccines, which can prevent typhoid in infants and young children, are being developed. Comparative immunogenicity studies are facilitated by an International Standard (IS) for human anti-Vi IgG. 16/138, a pool of sera from volunteers which received either Vi conjugate vaccine or plain Vi vaccine, was assessed as an IS alongside U.S. reference reagent Vi-IgGR1, 2011. Samples were tested in a commercial ELISA (n = 7), a standardised ELISA based on biotinylated Vi (n = 7) and in-house ELISAs (n = 7). Valid estimates were obtained for the potency of all samples in the commercial ELISA, and the commutability of 16/138 and Vi-IgGR1, 2011 was evident for the commercial ELISA and in-house ELISAs based on a coating of Vi and protein. The WHO Expert Committee on Biological Standardization established 16/138 as the first IS for anti-Vi IgG with 100 IU per ampoule and assigned 163 IU per vial of Vi-IgGR1, 2011.

Structure of a protective epitope of group B Streptococcus type III capsular polysaccharide

Despite substantial progress in the prevention of group B Streptococcus (GBS) disease with the introduction of intrapartum antibiotic prophylaxis, this pathogen remains a leading cause of neonatal infection. Capsular polysaccharide conjugate vaccines have been tested in phase I/II clinical studies, showing promise for further development. Mapping of epitopes recognized by protective antibodies is crucial for understanding the mechanism of action of vaccines and for enabling antigen design. In this study, we report the structure of the epitope recognized by a monoclonal antibody with opsonophagocytic activity and representative of the protective response against type III GBS polysaccharide. The structure and the atomic-level interactions were determined by saturation transfer difference (STD)-NMR and X-ray crystallography using oligosaccharides obtained by synthetic and depolymerization procedures. The GBS PSIII epitope is made by six sugars. Four of them derive from two adjacent repeating units of the PSIII backbone and two of them from the branched galactose-sialic acid disaccharide contained in this sequence. The sialic acid residue establishes direct binding interactions with the functional antibody. The crystal structure provides insight into the molecular basis of antibody-carbohydrate interactions and confirms that the conformational epitope is not required for antigen recognition. Understanding the structural basis of immune recognition of capsular polysaccharide epitopes can aid in the design of novel glycoconjugate vaccines.

Real-time PCR-based serotyping of Streptococcus agalactiae.

Group B Streptococcus (GBS) is an encapsulated, gram-positive pathogen that is an important cause of neonatal invasive infections, including sepsis and meningitis. There are ten known GBS serotypes based on distinct capsule compositions (Ia, Ib, II-IX), and current candidate capsular polysaccharide conjugate vaccines target only a subset of these. Serotyping of GBS isolates is important for understanding local epidemiology and for monitoring for serotype replacement or capsular switching. However, serotyping generally requires either latex agglutination, multiplex PCR with analysis of band sizes, or analysis of whole genome sequences–all techniques that are either expensive or not widely available. Here we report the development of a robust real-time PCR assay for determining GBS serotypes. Using both a diverse reference set of strains encompassing all ten serotypes and a collection of clinical isolates, we demonstrate concordance between real-time PCR serotyping and latex agglutination. We propose that real-time PCR serotyping represents an attractive alternative to current serotyping methods and may allow for improved acquisition of GBS serotype data.

Limited Efficacy of Antibacterial Vaccination Against Secondary Serotype 3 Pneumococcal Pneumonia Following Influenza Infection.

Secondary bacterial infections following influenza represent a major cause of mortality in the human population, which, in turn, has led to a call for stockpiling of bacterial vaccines for pandemic preparedness. To investigate the efficacy of bacterial vaccination for protection against secondary pneumococcal infection, mice were immunized with pneumococcal capsular polysaccharide conjugate vaccine, and then sequentially coinfected 5 weeks later with PR8 influenza virus and A66.1 Streptococcus pneumoniae. In the absence of influenza virus exposure, vaccination with polysaccharide conjugate vaccine was highly effective, as indicated by 100% survival from lethal pneumococcal pneumonia and 10 000-fold greater efficiency in clearance of bacteria from the lung compared to unvaccinated mice. Enhanced clearance after vaccination was dependent upon Fc receptor (FcR) expression. However, following influenza, <40% of vaccinated mice survived bacterial coinfection and FcR-dependent clearance of antibody-opsonized bacteria reduced bacterial levels in the lungs only 5-10 fold. No differences in lung myeloid cell numbers or in FcR cell surface expression were observed following influenza. The results show that induction of antibacterial humoral immunity is only partially effective in protection against secondary bacterial infections that occur following influenza, and suggest that additional therapeutic strategies to overcome defective antibacterial immunity should be explored.

Phase I clinical trial of O-acetylated pectin conjugate, a plant polysaccharide based typhoid vaccine

BackgroundTyphoid fever remains an important cause of morbidity and mortality in the developing countries. Vi capsular polysaccharide conjugate vaccine demonstrated safety and efficacy in young children in high endemic regions. A novel typhoid conjugate vaccine based on plant polysaccharide pectin was studied in a phase I trial. MethodsFruit pectin, having the same carbohydrate backbone structure as Vi, was purified from citrus peel and used as the polysaccharide source to prepare a semi-synthetic typhoid conjugate vaccine. Pectin was chemically O-acetylated (OAcPec) to antigenically resemble Vi and conjugated to carrier protein rEPA, a recombinant exoprotein A from Pseudomonas aeruginosa. 25 healthy volunteers, 18–45 years old, were injected once with OAcPec-rEPA. Safety and IgG antibodies reactive with Vi and pectin were analyzed. ResultsNo vaccine associated serious adverse reaction was reported. Six weeks after the injection of OAcPec-rEPA, 64% of the volunteers elicited >4-fold rise of anti-Vi IgG. At 26 weeks the level declined, but the difference between the levels at 6 and 26 weeks are not statistically significant. There is a direct correlation between the level of anti-Vi IgG before and after the injection (R2=0.96). The anti-Vi IgG can be absorbed by Vi, but not by pectin. There was no corresponding increase of anti-pectin after the injection, indicating the antibody response to OAcPec-rEPA was specific to Vi. There is no Vi-rEPA data in US adults for comparison of immune responses. The OAcPec-rEPA elicited significantly less IgG anti-Vi in US adults than those by Vi-rEPA in Vietnamese adults. ConclusionThe O-acetylated pectin conjugate, a plant based typhoid vaccine, is safe and immunogenic in adult volunteers.ClinicalTrial.gov identifier: NCT00277147, NIH Protocol ID number: OH06-CH-0070, FDA BB Investigation New Drug (IND) number 6989.

Nonprotective responses to pediatric vaccines occur in children who are otitis prone.

We recently found that children who experience recurrent otitis media despite individualized care (stringently-defined otitis prone [sOP]) do not develop an antibody response to several vaccine candidate protein antigens expressed by Streptococcus pneumonia (Spn) and Haemophilus influenzae. Here we sought to determine if these same children also failed to develop antibody to routine pediatric vaccinations. One hundred forty sera collected from children age 6-24 months were analyzed. sOP (n=34) and age-matched non-sOP (n=34) children were assessed for IgG concentrations to diphtheria toxoid, tetanus toxoid, pertussis toxoid, filamentous hemagglutinin, pertactin (DTaP), polio, hepatitis B, H. influenzae type b capsule polyribosyl-ribitol-phosphate (PRP) and Spn capsular polysaccharide conjugate vaccine. IgG protective titers to diphtheria toxoid (P=0.006), tetanus toxoid (P<0.0001), pertussis toxoid (P<0.0001), filamentous hemagglutinin (P=0.001), pertactin (P=0.005), hepatitis B (P<0.0001), polio 3 (P=0.03) and Spn 23F (P=0.01) but not polio 1,2, PRP or Spn 6B, and 14 were decreased in sOP versus non-sOP children using generalized estimating equations. A high percentage of sOP children had nonprotective antibody values that persisted until 24 months of age despite routine boosters. sOP children may fail to achieve protective antibody concentrations after several routine vaccinations.

Development of pneumococcal polysaccharide conjugate vaccine with long spacer arm

Streptococcus pneumoniae is a serious Gram-positive pathogen responsible for several life-threatening pneumococcal diseases. Pneumococcal capsular polysaccharide (CPS) is a key virulence determinant of S. pneumoniae and its immunogenicity can be improved by conjugation with a carrier protein. Reductive amination, the most widely used approach for pneumococcal CPS conjugate vaccine (PCV), suffers from low conjugation efficiency and the problem of steric hindrance. Here, copper-catalyzed azide-alkyne cycloaddition was used for development of PCV with long spacer arm (L-PCV). Tetanus toxoid (TT) was used as the carrier protein. The long spacer arm in L-PCV can minimize the problem of steric hindrance between CPS and TT, thereby improving the CPS-specific antibody titers in the mice model. L-PCV can also induce high avidity functional antibody and elicit immunological memory in response to the native CPS.

A bivalent vaccine to protect against Streptococcus pneumoniae and Salmonella typhi

Pneumococcal and Salmonella typhi infections are two major diseases for children in developing countries. For typhoid fever, licensed Vi polysaccharide vaccines are ineffective in children <2-year old. While investigational Vi conjugate vaccines have been shown effective in clinical trials, they are currently only available to restricted areas. Pneumococcal capsular polysaccharide conjugate vaccines are highly effective in children, but suffer from some limitations including cost and limited serotype coverage. We have previously shown that a fusion conjugate vaccine, consisting of pneumococcal fusion protein PsaA and pneumolysoid (PdT) conjugated to a polysaccharide, results in enhanced antibody and CD4+ Th17 cell responses as well as protection against pneumococcal colonization and disease in mice. Here we applied this approach to develop a bivalent vaccine against pneumococcus and S. typhi. Two species-conserved pneumococcal antigens (SP1572 or SP2070) were fused to the nonhemolytic pneumolysoid PdT. SP1572-PdT was then conjugated to Vi polysaccharide and SP2070-PdT was conjugated to the pneumococcal cell wall polysaccharide (CWPS; also conserved). Mice immunized with this bivalent conjugate were protected against pneumococcal colonization and sepsis challenges, and made anti-Vi antibody concentrations higher by 40-fold compared to mice that received equimolar mixtures of the antigens. An enhanced killing of Vi-bearing Salmonellae in vitro was demonstrated from plasma of mice that received the fusion conjugate but not the mixture of antigens. Our results support further evaluation of this bivalent immunogen for the prevention of pneumococcal colonization and disease, and of typhoid fever.

Intranasal immunization with recombinant PspA fused with a flagellin enhances cross-protective immunity against Streptococcus pneumoniae infection in mice

Streptococcus pneumoniae is a major respiratory pathogen that causes high levels of mortality and morbidity in infants and the elderly. Despite the use of antibiotics and vaccines, fatal pneumococcal disease remains prevalent. Pneumococcal surface protein A (PspA), a highly immunogenic surface protein produced by all strains of S. pneumoniae, can elicit protective immunity against fatal pneumococcal infection. We have previously demonstrated that the Vibrio vulnificus FlaB, a bacterial flagellin protein and agonist of TLR5, has strong mucosal adjuvant activity and induces protective immunity upon co-administration with tetanus toxoid. In this study, we have tested whether intranasal immunization with recombinant fusion proteins consisted of PspA and FlaB (PspA–FlaB and FlaB–PspA) is able to elicit more efficient protective mucosal immune responses against pneumococcal infection than immunization with PspA alone or with a stoichiometric mixture of PspA and FlaB. When mice were intranasally immunized with fusion proteins, significantly higher levels of anti-PspA IgG and IgA were induced in serum and mucosal secretions. The mice immunized intranasally with the FlaB–PspA fusion protein were the most protected from a lethal challenge with live S. pneumoniae, as compared to the mice immunized with PspA only, a mixture of PspA and FlaB, or the PspA–FlaB fusion protein. FlaB–PspA also induced a cross protection against heterologous capsular types. These results suggest that a FlaB–PspA fusion protein alone could be used as an anti-pneumococcal mucosal vaccine or as an effective partner protein for multivalent capsular polysaccharide conjugate vaccines.

Streptococcus pneumoniae Is Desiccation Tolerant and Infectious upon Rehydration

ABSTRACTStreptococcus pneumoniae (pneumococcus) is a frequent colonizer of the nasopharynx and one of the leading causative agents of otitis media, pneumonia, and meningitis. The current literature asserts that S. pneumoniae is transmitted person to person via respiratory droplets; however, environmental surfaces (fomites) have been linked to the spread of other respiratory pathogens. Desiccation tolerance has been to shown to be essential for long-term survival on dry surfaces. This study investigated the survival and infectivity of S. pneumoniae following desiccation under ambient conditions. We recovered viable bacteria after all desiccation periods tested, ranging from 1 h to 4 weeks. Experiments conducted under nutrient limitation indicate that desiccation is a condition separate from starvation. Desiccation of an acapsular mutant and 15 different clinical isolates shows that S. pneumoniae desiccation tolerance is independent of the polysaccharide capsule and is a species-wide phenomenon, respectively. Experiments demonstrating that nondesiccated and desiccated S. pneumoniae strains colonize the nasopharynx at comparable levels, combined with their ability to survive long-term desiccation, suggest that fomites may serve as alternate sources of pneumococcal infection.