Acute otitis media pneumococcal disease burden and nasopharyngeal colonization in children due to serotypes included and not included in current and new pneumococcal conjugate vaccines.

INTRODUCTION: Despite the introduction of effective pneumococcal conjugate vaccines (PCV), Streptococcus pneumoniae remains a major cause of acute otitis media (AOM) worldwide. New, higher valency vaccines that offer broader serotype coverage have been recently developed and others are in development. However, given the capsular serotypes expressed by pneumococci causing AOM, it is unclear to what extent differing or higher valency PCVs will provide additional protection. AREAS COVERED: We conducted a systematic literature search of the MEDLINE database to identify articles published from January 2016 to September 2021 in 4 low and middle income and 10 high-income countries. We searched PubMed with terms: (Streptococcus pneumoniae) OR pneumococcal AND serotype AND (conjugate vaccine). We evaluated serotype distribution and the actual or projected coverage of pneumococcal serotypes by PCV10 (GlaxoSmithKline), PCV13 (Pfizer), PCV10SII (Serum Institute of India) PCV15 (Merck) and PCV20 (Pfizer). EXPERT OPINION: Our review highlights the important epidemiological differences in serotype distribution and coverage by existing and higher valency vaccines to protect against AOM in children. These data provide support for further evaluation of serotype-independent vaccines for optimal control of pneumococcal AOM disease worldwide.

A Phase 1 Randomized, Placebo-controlled, Observer-blinded Trial to Evaluate the Safety and Immunogenicity of Inactivated Streptococcus pneumoniae Whole-cell Vaccine in Adults.

BACKGROUND: Broadly protective pneumococcal vaccines that are affordable for low-resource countries are needed. Streptococcus pneumoniae whole cell vaccine (wSp) is an investigational vaccine that contains killed cells from a nonencapsulated strain of S. pneumoniae (SPn) with aluminum hydroxide adjuvant. Studies in mice demonstrated protection against nasopharyngeal carriage (T-cell-mediated) and invasive pneumococcal disease (antibody-mediated). The aim of this randomized, double-blind, placebo-controlled Phase 1 study was to assess safety, tolerability and immunogenicity of wSp in healthy adults. METHODS: Forty-two participants were randomized into 3 dose cohorts to receive 0.1, 0.3, or 0.6 mg of wSp or saline intramuscularly. Participants received a 3-dose vaccination schedule spaced by 4-week intervals. Postvaccination assessments included solicited reactogenicity events through day 7, blood chemistry and hematology assessments at day 7, and adverse events (AEs) through day 84. Participants were monitored for serum antibody and peripheral blood mononuclear cell cytokine responses to pneumococcal antigens. A 6-month telephone follow-up was completed to assess for any additional AEs. RESULTS: wSp was safe and well tolerated. Reactogenicity was acceptable and no untoward safety signals were observed. wSp elicited potentially clinically significant rises (defined arbitrarily as at least a 2-fold rise) in immunoglobulin G responses to multiple pneumococcal antigens, including pneumococcal surface protein A and pneumolysin. Functional antibody responses were observed with the highest dose of wSp (0.6 mg). Increases in T-cell cytokine responses, including interleukin 17A, were also seen among wSp vaccines. CONCLUSIONS: wSp was safe and well tolerated in healthy US adults, eliciting pneumococcal antigen-specific antibody and T-cell cytokine responses.

Development of a whole cell pneumococcal vaccine: BPL inactivation, cGMP production, and stability.

Pneumococcal infections impose a large burden of disease on the human population, mainly in developing countries, and the current pneumococcal vaccines offer serotype-specific protection, but do not cover all pathogenic strains, leaving populations vulnerable to disease caused by non-vaccine serotypes. The pneumococcal whole cell vaccine is a low-cost strategy based on non-capsular antigens common to all strains, inducing serotype-independent immunity. Therefore, we developed the process for the cGMP production of this cellular vaccine. Initially, three engineering runs and two cGMP runs were performed in 60-L bioreactors, demonstrating the consistency of the production process, as evaluated by the growth curves, glucose consumption and metabolite formation (lactate and acetate). Cell recovery by tangential filtration was 92 ± 13 %. We optimized the conditions for beta-propiolactone (BPL) inactivation of the bacterial suspensions, establishing a maximum cell density of OD600 between 27 and 30, with a BPL concentration of 1:4000 (v/v) at 150 rpm and 4 °C for 30 h. BPL was hydrolyzed by heating for 2h at 37 °C. The criteria and methods for quality control were defined using the engineering runs and the cGMP Lots passed all specifications. cGMP vaccine Lots displayed high potency, inducing between 80 and 90% survival in immunized mice when challenged with virulent pneumococci. Sera from mice immunized with the cGMP Lots recognized several pneumococcal proteins in the extract of encapsulated strains by Western blot. The cGMP whole cell antigen bulk and whole cell vaccine product lots were shown to be stable for up to 12 and 18 months, respectively, based upon survival assays following i.p. challenge. Our results show the consistency and stability of the cGMP whole cell pneumococcal vaccine lots and demonstrate the feasibility of production in a developing country setting.

Leaning in to the power of the possible: the crucial role of women scientists on preventing Haemophilus influenzae type b disease.

Beginning in an era when female scientists were a lonely minority, women have made major contributions to our understanding of Haemophilus influenzae type b (Hib) as a pathogen, its treatment and its prevention. The individual scientific and public health contributions, and their collective impact, are reviewed in the context of the development and successful implementation of highly efficacious Hib vaccines that are now being deployed to nearly every country worldwide for the prevention of life-threatening pediatric Hib disease.

Broad antibody and T cell reactivity induced by a pneumococcal whole-cell vaccine.

Injecting mice with killed cells of non-capsulated strain RM200 adsorbed on Al(OH)3 (pneumococcal whole-cell vaccine; WCV) reduces nasopharyngeal colonization by capsular serotype 6B and prevents fatal aspiration pneumonia by serotype 3 or serotype 5 strains. To further examine the potential for omni-strain immunity, we here examined a panel of clinical isolates and a library of capsule-switch variants in the TIGR4 background. IgG binding to these bacteria in sera of rabbits injected with WCV or Al(OH)3 alone was assayed by ELISA without and with adsorption with cell-wall polysaccharide, a species-common antigen. The examined strains were 23 primary isolates including at least 10 different MLS types and 13 serotypes; 15 of these strains were invasive isolates, subsequently mouse-passed. Additionally, to investigate the effect of capsulation, TIGR4 strain constructs with the capsulation genes of 20 different serotypes were evaluated. In ELISA all strains showed a large difference in IgG binding due to the immunization, of which most of the antibody typically was not CWPS-adsorbed and presumably directed to exposed protein antigens. Increased binding of IgG in the WCV-immunized serum to the 20 isogenic capsule-switch strains was shown also by flow cytometry. Further, all these 20 strains elicited IL-17A in T cells of WCV-vaccinated mice, a cytokine known to accelerate pneumococcal clearance. Thus WCV induced both humoral and T(H)17 cell-mediated immunity against all tested strains.

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.

Serotype-independent pneumococcal experimental vaccines that induce cellular as well as humoral immunity.

For prevention of Streptococcus pneumoniae (pneumococcus) infections in infancy, protein-conjugated capsular polysaccharide vaccines provide serotype-specific, antibody-mediated immunity but do not cover all of the 90+ capsule serotypes. Therefore, microbiologists have sought protective noncapsular antigens common to all strains. Alternatively, we investigated killed cells of a noncapsulated strain, which expose many such common antigens. Given to mice intranasally, this vaccine elicits antibody-independent, CD4+ T lymphocyte-dependent accelerated clearance of pneumococci of various serotypes from the nasopharynx mediated by the cytokine IL-17A. Such immunity may reproduce the natural resistance that develops in infants before capsular antibodies arise. Given by injection, the killed cell vaccine induces bifunctional immunity: plasma antibodies protective against fatal pneumonia challenge, as well as IL-17A-mediated nasopharyngeal clearance. Human testing of this inexpensive candidate vaccine by intramuscular injection is planned. Bacterial cellular vaccines are complex--a challenge for reproducibility. However, when several known protective antigens were deleted, the killed pneumococcal vaccine was still protective. This antigenic redundancy may prevent vaccine escape variants by recombinational loss, which is frequent in pneumococcus. Biochemically defined immunogens with bifunctional activity have also been devised. These immunogens are three-component conjugates in which cell wall teichoic acid (a common antigen capable of T cell activation) is coupled to a genetic fusion of two common pneumococcal proteins: a protective surface antigen and a derivative of pneumolysin, which provides TLR4 agonist activity and induces antitoxic immunity. Such constructs induce accelerated clearance when given intranasally and induce both immune mechanisms when injected. The defined composition permits analysis of structure-function activity.

GMP-grade pneumococcal whole-cell vaccine injected subcutaneously protects mice from nasopharyngeal colonization and fatal aspiration-sepsis.

Mucosal immunization with a killed whole-cell pneumococcal vaccine, given with enterotoxin-related adjuvants, has been shown to confer multi-serotype protection against colonization of the nasopharynx and middle ear in mice. However, because novel mucosal immunization strategies may be difficult to implement, here we evaluated subcutaneous injection. Strain RM200 was engineered to be capsule-negative, autolysin-negative, and to express a non-toxic mutant pneumolysoid. Liter-scale and 60-l Good Manufacturing Practice (GMP) cultures were grown in bovine-free soy-based medium, killed with chloroform or beta-propiolactone, and injected into C57Bl/6 mice without or with aluminum adjuvant. The adjuvant Al(OH)(3) strongly increased responses, particularly if pre-treated with phosphate. Protection was found in several tested model infections: nasal colonization with a serotype 6B strain and fatal aspiration-sepsis with strains of serotype 3 and 5. Protection against colonization was mechanistically dependent on the presence of CD4+ T cells at the time of challenge; in contrast, in the type 3 aspiration-sepsis model, CD4+ T cells were not required for protection at the time of challenge, suggesting that antibody alone was sufficient to protect against death in this model. Rabbits receiving sequential intramuscular injections in a pilot toxicity study displayed local reactogenicity at injection sites but no clinical signs. The rabbit antiserum thus produced was active in an in vitro phagocytic killing assay and passively protected mice in the type 3 aspiration-sepsis model. Approval is being sought for human trials of this vaccine.

Options for inactivation, adjuvant, and route of topical administration of a killed, unencapsulated pneumococcal whole-cell vaccine.

We previously reported that ethanol-killed cells of a noncapsulated strain of Streptococcus pneumoniae, given intranasally with cholera toxin as an adjuvant, protect rats against pneumonia and mice against colonization of the nasopharynx and middle ear by capsulated pneumococci of various serotypes. The acceleration of pneumococcal clearance from the nasopharynx in mice is CD4+ T cell-dependent and interleukin 17A (IL-17A) mediated and can be antibody independent. Here, anticipating human studies, we have demonstrated protection with a new vaccine strain expressing a nonhemolytic derivative of pneumolysin and grown in bovine-free culture medium. Killing the cells with chloroform, trichloroethylene, or beta-propiolactone--all used without postinactivation washing--produced more-potent immunogens than ethanol, and retention of soluble components released from the cells contributed to protection. Two sequential intranasal administrations of as little as 1 microg of protein (total of cellular and soluble combined) protected mice against nasopharyngeal challenge with pneumococci. Nontoxic single and double mutants of Escherichia coli heat-labile toxin were effective as mucosal adjuvants. Protection was induced by the sublingual and buccal routes, albeit requiring larger doses than when given intranasally. Protection was likewise induced transdermally with sonicates of the killed-cell preparation. Thus, this whole-cell antigen can be made and administered in a variety of ways to suit the manufacturer and the vaccination program and is potentially a solution to the need for a low-cost vaccine to reduce the burden of childhood pneumococcal disease in low-income countries.

GMP-grade pneumococcal whole-cell vaccine injected subcutaneously protects mice from nasopharyngeal colonization and fatal aspiration-sepsis

Mucosal immunization with a killed whole-cell pneumococcal vaccine, given with enterotoxin-related adjuvants, has been shown to confer multi-serotype protection against colonization of the nasopharynx and middle ear in mice. However, because novel mucosal immunization strategies may be difficult to implement, here we evaluated subcutaneous injection. Strain RM200 was engineered to be capsule-negative, autolysin-negative, and to express a non-toxic mutant pneumolysoid. Liter-scale and 60-l Good Manufacturing Practice (GMP) cultures were grown in bovine-free soy-based medium, killed with chloroform or beta-propiolactone, and injected into C57Bl/6 mice without or with aluminum adjuvant. The adjuvant Al(OH)3 strongly increased responses, particularly if pre-treated with phosphate. Protection was found in several tested model infections: nasal colonization with a serotype 6B strain and fatal aspiration-sepsis with strains of serotype 3 and 5. Protection against colonization was mechanistically dependent on the presence of CD4+ T cells at the time of challenge; in contrast, in the type 3 aspiration-sepsis model, CD4+ T cells were not required for protection at the time of challenge, suggesting that antibody alone was sufficient to protect against death in this model. Rabbits receiving sequential intramuscular injections in a pilot toxicity study displayed local reactogenicity at injection sites but no clinical signs. The rabbit antiserum thus produced was active in an in vitro phagocytic killing assay and passively protected mice in the type 3 aspiration-sepsis model. Approval is being sought for human trials of this vaccine.

Mechanisms in the serotype-independent pneumococcal immunity induced in mice by intranasal vaccination with the cell wall polysaccharide.

We previously reported that cell wall polysaccharide (CWPS) given to mice intranasally with adjuvant induces serotype-independent immunity to pneumococci. Some strains make CWPS with one phosphocholine group (CWPS/1), but most express two per tetrasaccharide repeat unit (CWPS/2). Here, CWPS/1 and CWPS/2 were equally protective against colonization by CWPS/2-type pneumococci, but the related Streptococcus mitis polymer lacking phosphocholine was non-protective. Previously the protection was shown to be CD4+T cell-dependent, abrogated by antiserum to interleukin (IL)-17A, and demonstrable in antibody-defective mice. Here, CWPS failed to protect IL-17A receptor knockout mice, further indicating IL-17A-dependence. When commercial CWPS/1 was size-fractionated preparatively, the larger exceeded the smaller molecules in their capacity to prime for IL-17A responses, and only the larger protected against pneumococcal colonization. However, a CWPS-tetanus toxoid conjugate - despite raising high titers of phosphocholine antibody - was non-protective, confirming the irrelevance of humoral immunity in this model. The results strengthen the concept that IL-17A-mediated T cell immunity is inducible by zwitterionic polysaccharides with sufficient chain length to provide coiled secondary structure. Coupling CWPS to protein, which paradoxically prevents protection, may occlude this regular linear conformation. We suggest that mucosal immunization with CWPS primes T(H)17 cells, which - upon contact with the phosphocholine of colonizing pneumococci - elaborate IL-17A, enhancing phagocytosis.

Pneumococcal capsular polysaccharide structure predicts serotype prevalence.

There are 91 known capsular serotypes of Streptococcus pneumoniae. The nasopharyngeal carriage prevalence of particular serotypes is relatively stable worldwide, but the host and bacterial factors that maintain these patterns are poorly understood. Given the possibility of serotype replacement following vaccination against seven clinically important serotypes, it is increasingly important to understand these factors. We hypothesized that the biochemical structure of the capsular polysaccharides could influence the degree of encapsulation of different serotypes, their susceptibility to killing by neutrophils, and ultimately their success during nasopharyngeal carriage. We sought to measure biological differences among capsular serotypes that may account for epidemiological patterns. Using an in vitro assay with both isogenic capsule-switch variants and clinical carriage isolates, we found an association between increased carriage prevalence and resistance to non-opsonic neutrophil-mediated killing, and serotypes that were resistant to neutrophil-mediated killing tended to be more heavily encapsulated, as determined by FITC-dextran exclusion. Next, we identified a link between polysaccharide structure and carriage prevalence. Significantly, non-vaccine serotypes that have become common in vaccinated populations tend to be those with fewer carbons per repeat unit and low energy expended per repeat unit, suggesting a novel biological principle to explain patterns of serotype replacement. More prevalent serotypes are more heavily encapsulated and more resistant to neutrophil-mediated killing, and these phenotypes are associated with the structure of the capsular polysaccharide, suggesting a direct relationship between polysaccharide biochemistry and the success of a serotype during nasopharyngeal carriage and potentially providing a method for predicting serotype replacement.

Protection against Pneumococcal colonization and fatal pneumonia by a trivalent conjugate of a fusion protein with the cell wall polysaccharide.

Cell wall polysaccharide (CWPS), pneumolysin, and surface adhesin A (PsaA) are antigens common to virtually all serotypes of Streptococcus pneumoniae (pneumococcus), and all have been studied separately for use in protection. Previously we showed that protection against nasopharyngeal (NP) colonization by intranasal vaccination of mice with killed pneumococci is mediated by T(H)17 cells and correlates with interleukin-17A (IL-17A) expression by T cells in vitro; we have also shown that CWPS and other species-common antigens protect against colonization by a similar mechanism. Here we made a fusion protein of PsaA with the pneumolysin nontoxic derivative PdT and then coupled CWPS to the fusion protein, aiming to enhance immune responses to all three antigens. When given intranasally with cholera toxin adjuvant, the fusion conjugate induced higher serum antibody titers and greater priming for IL-17A responses than an equimolar mixture of the three antigens. The conjugate administered intranasally protected mice against experimental NP colonization by a strain of serotype 6B, while mice immunized with the mixture or with bivalent conjugates were not protected. Subcutaneous immunization with the conjugate and alum adjuvant likewise induced higher antibody titers than the mixture, primed for IL-17A responses, and reduced colonization. The conjugate, but not the antigen mixture, fully protected mice from fatal pneumonia caused by a highly virulent serotype 3 strain. Thus, a covalent construct of three antigens common to all serotypes exhibits protection with both mucosal and systemic administration.

Interleukin-17A mediates acquired immunity to pneumococcal colonization.

Although anticapsular antibodies confer serotype-specific immunity to pneumococci, children increase their ability to clear colonization before these antibodies appear, suggesting involvement of other mechanisms. We previously reported that intranasal immunization of mice with pneumococci confers CD4+ T cell-dependent, antibody- and serotype-independent protection against colonization. Here we show that this immunity, rather than preventing initiation of carriage, accelerates clearance over several days, accompanied by neutrophilic infiltration of the nasopharyngeal mucosa. Adoptive transfer of immune CD4+ T cells was sufficient to confer immunity to naïve RAG1(-/-) mice. A critical role of interleukin (IL)-17A was demonstrated: mice lacking interferon-gamma or IL-4 were protected, but not mice lacking IL-17A receptor or mice with neutrophil depletion. In vitro expression of IL-17A in response to pneumococci was assayed: lymphoid tissue from vaccinated mice expressed significantly more IL-17A than controls, and IL-17A expression from peripheral blood samples from immunized mice predicted protection in vivo. IL-17A was elicited by pneumococcal stimulation of tonsillar cells of children or adult blood but not cord blood. IL-17A increased pneumococcal killing by human neutrophils both in the absence and in the presence of antibodies and complement. We conclude that IL-17A mediates pneumococcal immunity in mice and probably in humans; its elicitation in vitro could help in the development of candidate pneumococcal vaccines.

Antibody-independent, interleukin-17A-mediated, cross-serotype immunity to pneumococci in mice immunized intranasally with the cell wall polysaccharide.

Serotype-specific immunity to Streptococcus pneumoniae is conferred by antibodies to the capsular polysaccharides, which define the 90 known serotypes. Whether antibody to the species-common cell wall polysaccharide (C-Ps) is protective has been a matter of controversy. Here we show that C-Ps given intranasally with mucosal adjuvant increased the resistance of mice to experimental nasopharyngeal colonization by capsulated S. pneumoniae of serotype 6B. This immunity could be induced in mice congenitally lacking immunoglobulin but was dependent upon CD4+ T cells. Elimination of the charged amino group on the polymer backbone by N acetylation of C-Ps reduced the immunity, as did treatment of the mice with antibody to the cytokine interleukin-17A at the time of challenge, both consistent with the hypothesis of T-cell activation due to the zwitterionic motif of the polymer. C-Ps also protected in a model of fatal aspiration pneumonia by heavily capsulated serotype 3. These findings suggest a novel immunization strategy against S. pneumoniae.

CD4+ T cells mediate antibody-independent acquired immunity to pneumococcal colonization.

Acquired immunity to Streptococcus pneumoniae (pneumococcus) has long been assumed to depend on the presence of anticapsular antibodies. We found, however, that colonization with live pneumococci of serotypes 6B, 7F, or 14 protected mice against recolonization by any of the serotypes and that protection from acquisition of a heterologous or homologous strain did not depend on anticapsular antibody. Further, intranasal immunization by live pneumococcal colonization or by a killed, nonencapsulated whole-cell vaccine protected antibody-deficient mice against colonization, suggesting independence of antibodies to any pneumococcal antigens. Protection by intranasal immunization with whole-cell vaccine was completely abrogated in T cell-deficient mice, and in mice that were congenitally deficient in CD4(+) T cells or depleted of these cells at the time of challenge. In contrast, mice congenitally deficient in, or depleted of, CD8(+) T cells were fully protected. Protection in this model was observed beyond 2 months after immunization, arguing against innate or nonspecific immune mechanisms. Thus, we find that immunity to pneumococcal colonization can be induced in the absence of antibody, independent of the capsular type, and this protection requires the presence of CD4(+) T cells at the time of challenge.

Multiserotype protection of mice against pneumococcal colonization of the nasopharynx and middle ear by killed nonencapsulated cells given intranasally with a nontoxic adjuvant.

Intranasal challenge of C57BL/6 mice with Streptococcus pneumoniae serotypes 6B, 14, and 23F produced colonization of the middle ear and NP. Intranasal vaccination with ethanol-killed nonencapsulated cells with adjuvant protected both sites. Of four nontoxic adjuvants tested, the cholera toxin B subunit was most effective and least nonspecifically protective.

Non-interference between two protein carriers when used with the same polysaccharide for pneumococcal conjugate vaccines in 2-year-old children.

The carriers tetanus toxoid (T) and diphtheria CRM-197 (C) were compared in conjugate vaccines using identical coupling chemistry and polysaccharide (PS) loading, for safety and immunogenicity in 2-year-old children. Also tested were a mixture of halved doses of both carriers bearing the same PS serotypes. For this study, PS types 6A, 14, 19F, and 23F (separately) were coupled to T or C by reductive amination at PS/protein ratios of 0.50+/-0.18. With each carrier the four PS types were combined, giving the tetravalent vaccines "T-6, -14, -19, -23" or "C-6, -14, -19, -23" containing 50 microg of the carrier and roughly 20 microg total PS per ml of saline (no adjuvant). The children received primary (1') injections of 50 microg (protein) of either vaccine or a mixture of 25 microg of both; identical secondary (2') injections were given 2 months afterwards. Sera were taken before the 1' and 2' and 1 month post-2', and serum IgG responses to the four PS were determined by ELISA. For geometric mean (GM) post-1' antibody, "C-6, -14, -19, -23" exceeded "T-6, -14, -19, -23" for type 19F; for 2' antibody, "T-6, -14, -19, -23" exceeded "C-6, -14, -19, -23" for type 14, but "C-6, -14, -19, -23" and the T/C mixture exceeded "T-6, -14, -19, -23" for the type 23F response. No other differences were significant. Analyzed by individual fold-rises, "C-6, -14, -19, -23" and the T/C mixture exceeded "T-6, -14, -19, -23" for types 19F and 23F. Thus, there was no consistent difference between the T and C carriers; rather, the results differed by serotype. When a mixture of halved doses of "T-6, -14, -19, -23" and "C-6, -14, -19, -23" was injected, neither negative nor positive interference with the PS antibody responses was found. Anticipating multivalent PS conjugate vaccines of the future to be used in infancy, this strategy would have two hypothetical advantages worth further investigation-avoiding "carrier epitopic overload" by reducing each carrier dosage and recruiting T-helper activity by both carriers for each PS.