Variable BCG efficacy in rhesus populations: Pulmonary BCG provides protection where standard intra-dermal vaccination fails.

M.bovis BCG vaccination against tuberculosis (TB) notoriously displays variable protective efficacy in different human populations. In non-human primate studies using rhesus macaques, despite efforts to standardise the model, we have also observed variable efficacy of BCG upon subsequent experimental M. tuberculosis challenge. In the present head-to-head study, we establish that the protective efficacy of standard parenteral BCG immunisation varies among different rhesus cohorts. This provides different dynamic ranges for evaluation of investigational vaccines, opportunities for identifying possible correlates of protective immunity and for determining why parenteral BCG immunisation sometimes fails. We also show that pulmonary mucosal BCG vaccination confers reduced local pathology and improves haematological and immunological parameters post-infection in animals that are not responsive to induction of protection by standard intra-dermal BCG. These results have important implications for pulmonary TB vaccination strategies in the future.

Protection Induced by Simultaneous Subcutaneous and Endobronchial Vaccination with BCG/BCG and BCG/Adenovirus Expressing Antigen 85A against Mycobacterium bovis in Cattle.

The incidence of bovine tuberculosis (bTB) in the GB has been increasing since the 1980s. Immunisation, alongside current control measures, has been proposed as a sustainable measure to control bTB. Immunisation with Mycobacterium bovis bacillus Calmette-Guerin (BCG) has been shown to protect against bTB. Furthermore, much experimental data indicates that pulmonary local immunity is important for protection against respiratory infections including Mycobacterium tuberculosis and that pulmonary immunisation is highly effective. Here, we evaluated protection against M. bovis, the main causative agent of bTB, conferred by BCG delivered subcutaneously, endobronchially or by the new strategy of simultaneous immunisation by both routes. We also tested simultaneous subcutaneous immunisation with BCG and endobronchial delivery of a recombinant type 5 adenovirus expressing mycobacterial antigen 85A. There was significantly reduced visible pathology in animals receiving the simultaneous BCG/BCG or BCG/Ad85 treatment compared to naïve controls. Furthermore, there were significantly fewer advanced microscopic granulomata in animals receiving BCG/Ad85A compared to naive controls. Thus, combining local and systemic immunisation limits the development of pathology, which in turn could decrease bTB transmission.

A novel murine cytomegalovirus vaccine vector protects against Mycobacterium tuberculosis.

Tuberculosis remains a global health problem so that a more effective vaccine than bacillus Calmette-Guérin is urgently needed. Cytomegaloviruses persist lifelong in vivo and induce powerful immune and increasing ("inflationary") responses, making them attractive vaccine vectors. We have used an m1-m16-deleted recombinant murine CMV (MCMV) expressing Mycobacterium tuberculosis Ag 85A to show that infection of mice with this recombinant significantly reduces the mycobacterial load after challenge with M. tuberculosis, whereas control empty virus has a lesser effect. Both viruses induce immune responses to H-2(d)-restricted epitopes of MCMV pp89 and M18 Ags characteristic of infection with other MCMVs. A low frequency of 85A-specific memory cells could be revealed by in vivo or in vitro boosting or after challenge with M. tuberculosis. Kinetic analysis of M. tuberculosis growth in the lungs of CMV-infected mice shows early inhibition of M. tuberculosis growth abolished by treatment with NK-depleting anti-asialo ganglio-N-tetraosylceramide Ab. Microarray analysis of the lungs of naive and CMV-infected mice shows increased IL-21 mRNA in infected mice, whereas in vitro NK assays indicate increased levels of NK activity. These data indicate that activation of NK cells by MCMV provides early nonspecific protection against M. tuberculosis, potentiated by a weak 85A-specific T cell response, and they reinforce the view that the innate immune system plays an important role in both natural and vaccine-induced protection against M. tuberculosis.

Pneumococcal conjugate and plain polysaccharide vaccines have divergent effects on antigen-specific B cells.

BACKGROUND: A 23-valent unconjugated pneumococcal polysaccharide vaccine (23vP), routinely administered at the age of 65, has limited effectiveness, and revaccination induces attenuated antibody responses. It is not known whether pneumococcal polysaccharide-protein conjugated vaccines (PCV), although highly effective in infants, offer any immunological advantages over 23vP in adults. METHODS: We immunized adults with schedules combining both PCV and 23vP and investigated B-cell responses to establish whether PCV7 (a 7-valent PCV) induced T-dependent responses in adults, to assess the role of memory B cells in 23vP-induced antibody hyporesponsiveness, and to identify the B-cell subtypes involved. RESULTS: A single dose of PCV7 induced significant increases in serotype-specific memory B-cell populations in peripheral blood indicating a T-dependent response. Conversely, immunization with 23vP resulted in a decrease in memory B-cell frequency. Furthermore, memory B-cell responses to subsequent immunization with PCV7, when given after 23vP, were attenuated. Notably, B1b cells, a subset important in protecting mice against pneumococci, were also depleted following immunization with 23vP in humans. CONCLUSIONS: This study indicates that PCV7 may have an immunological advantage over 23vP in adults and that 23vP-induced depletion of memory and B1b-cell subsets may provide a basis for antibody hyporesponsiveness and the limited effectiveness of 23vP. Clinical Trials Registration. ISRCTN: 78768849.

Simultaneous immunization against tuberculosis.

BACKGROUND: BCG, the only licensed vaccine against tuberculosis, provides some protection against disseminated disease in infants but has little effect on prevention of adult pulmonary disease. Newer parenteral immunization prime boost regimes may provide improved protection in experimental animal models but are unproven in man so that there remains a need for new and improved immunization strategies. METHODS AND FINDINGS: Mice were immunized parenterally, intranasally or simultaneously by both routes with BCG or recombinant mycobacterial antigens plus appropriate adjuvants. They were challenged with Mycobacterium tuberculosis (Mtb) and the kinetics of Mtb growth in the lungs measured. We show that simultaneous immunization (SIM) of mice by the intranasal and parenteral routes is highly effective in increasing protection over parenteral BCG administration alone. Intranasal immunization induces local pulmonary immunity capable of inhibiting the growth of Mtb in the early phase (the first week) of infection, while parenteral immunization has a later effect on Mtb growth. Importantly, these two effects are additive and do not depend on priming and boosting the immune response. The best SIM regimes reduce lung Mtb load by up to 2 logs more than BCG given by either route alone. CONCLUSIONS: These data establish SIM as a novel and highly effective immunization strategy for Mtb that could be carried out at a single clinic visit. The efficacy of SIM does not depend on priming and boosting an immune response, but SIM is complementary to prime boost strategies and might be combined with them.

CXCR6 is a marker for protective antigen-specific cells in the lungs after intranasal immunization against Mycobacterium tuberculosis.

Convincing correlates of protective immunity against tuberculosis have been elusive. In BALB/c mice, intranasal immunization with a replication-deficient recombinant adenovirus expressing Mycobacterium tuberculosis antigen 85A (adenovirus-85A) induces protective lower respiratory tract immunity against pulmonary challenge with Mycobacterium tuberculosis, while intradermal immunization with adenovirus-85A does not. Here we report that intranasal immunization with adenovirus-85A induces expression of the chemokine receptor CXCR6 on lung CD8 T lymphocytes, which is maintained for at least 3 months. CXCR6-positive antigen-specific T cell numbers are increased among bronchoalveolar lavage-recoverable cells. Similarly, intranasal immunization with recombinant antigen 85A with adjuvant induces CXCR6 expression on lung CD4 cells in BALB/c and C57BL/6 mice, while a synthetic ESAT6(1-20) peptide with adjuvant induces CXCR6 expression in C57BL/6 mice. Parenteral immunization fails to do so. Upregulation of CXCR6 is accompanied by a transient elevation of serum CXCL16 after intranasal immunization, and lung cells cultured ex vivo from mice immunized intranasally show increased production of CXCL16. Administration of CXCL16 and cognate antigen intranasally to mice previously immunized parenterally increases the number of antigen-specific T lymphocytes in the bronchoalveolar lavage-recoverable population, which mediates inhibition of the early growth of Mycobacterium tuberculosis after challenge. We conclude that expression of CXCR6 on lung T lymphocytes is a correlate of local protective immunity against Mycobacterium tuberculosis after intranasal immunization and that CXCR6 and CXCL16 play an important role in the localization of T cells within lung tissue and the bronchoalveolar lavage-recoverable compartment.

A randomized study comparing combined pneumococcal conjugate and polysaccharide vaccination schedules in adults.

BACKGROUND: The widely used 23-valent plain polysaccharide vaccine (23vP) has limited effectiveness, produces short-lived immune responses, and induces attenuated antibody production after subsequent challenge with pneumococcal vaccines. Our goal was to examine whether priming with the 7-valent pneumococcal conjugate vaccine (PCV7) could enhance the immunogenicity of 23vP for the PCV7 serotypes and to investigate whether 23vP induced hyporesponsiveness could be overcome using PCV7. METHODS: We conducted an open-label randomized study that compared 3 vaccine schedules, each of which consisted of 2 doses of PCV7 and 1 dose of 23vP (23vP-PCV7-PCV7, PCV7-23vP-PCV7, PCV7-PCV7-23vP) administered over a 1-year period in a cohort of 348 adults 50-70 years of age. All vaccines were administered intramuscularly and were given 6 months apart. Blood samples were obtained prior to and 1 month after each vaccination. RESULTS: 23vP administered after priming with 2 doses of PCV7 produced significantly higher antibody concentrations for 3 of the 7 PCV7 serotypes, compared with vaccination with a single dose of 23vP; however, the same immunogenicity could be achieved with a single dose of PCV7. Prior vaccination with 23vP attenuated the antibody response to subsequent PCV7, which was not restored by additional doses of PCV7. CONCLUSION: In adults, vaccination schedules combining PCV7 and 23vP do not provide improved immunogenicity over the use of a single dose of 23vP for most of the serotypes contained in PCV7.

Human cytomegalovirus-specific CD8(+) T-cell expansions contain long-lived cells that retain functional capacity in both young and elderly subjects.

The immune response to human cytomegalovirus (HCMV) infection is characterized by the accumulation of HCMV-specific CD8(+) T cells, particularly in the elderly; such expansions may impair immune responses to other pathogens. We investigated mechanisms underlying HCMV-specific expansions in 12 young and 21 old healthy subjects (although not all analyses were performed on all subjects). Phenotypically, HCMV-pentamer(+) CD8(+) T cells were characterized by marked Vß restriction, advanced differentiation (being predominantly CD27(-) CD28(-) ), and variable CD45RO/RA expression. Although more common and larger in older subjects, expansions had similar phenotypic characteristics in the young. In one old subject, repeated studies demonstrated stability in size and Vß distribution of pentamer(+) populations over 6 years. We tested whether HCMV-specific CD8(+) T-cell expansions arose from accelerated proliferation or extended lifespan by in vivo labelling with deuterated glucose and ex vivo Ki-67 expression. Uptake of deuterated glucose was lower in pentamer(+) cells than in pentamer(-) CD8(+) CD45RO(+) or CD8(+) CD45RA(+) cells in three old subjects, consistent with reduced proliferation and extended lifespan. Similarly Ki-67 labelling showed no evidence for increased proliferation in HCMV-specific CD8(+) expansions in older subjects, although pentamer(-) CD45RA(+) cells from young donors expressed very little Ki-67. We investigated Bcl-2 and CD95 as possible anti-apoptotic mediators, but neither was associated with pentamer-positivity. To investigate whether expansion represents a compensatory response to impaired functionality, we performed two tests of functionality, peptide-stimulated proliferation and CD107 expression; both were intact in pentamer(+) cells. Our data suggest that HCMV-specific CD8(+) expansions in older subjects accumulate by extended lifespan, rather than accelerated proliferation.

Chemokine gene expression in lung CD8 T cells correlates with protective immunity in mice immunized intra-nasally with Adenovirus-85A.

BACKGROUND: Immunization of BALB/c mice with a recombinant adenovirus expressing Mycobacterium tuberculosis (M. tuberculosis) antigen 85A (Ad85A) protects against aerosol challenge with M. tuberculosis only when it is administered intra-nasally (i.n.). Immunization with Ad85A induces a lung-resident population of activated CD8 T cells that is antigen dependent, highly activated and mediates protection by early inhibition of M. tuberculosis growth. In order to determine why the i.n. route is so effective compared to parenteral immunization, we used microarray analysis to compare gene expression profiles of pulmonary and splenic CD8 T cells after i.n. or intra-dermal (i.d.) immunization. METHOD: Total RNA from CD8 T cells was isolated from lungs or spleens of mice immunized with Ad85A by the i.n. or i.d. route. The gene profiles generated from each condition were compared. Statistically significant (p ≤ 0.05) differentially expressed genes were analyzed to determine if they mapped to particular molecular functions, biological processes or pathways using Gene Ontology and Panther DB mapping tools. RESULTS: CD8 T cells from lungs of i.n. immunized mice expressed a large number of chemokines chemotactic for resting and activated T cells as well as activation and survival genes. Lung lymphocytes from i.n. immunized mice also express the chemokine receptor gene Cxcr6, which is thought to aid long-term retention of antigen-responding T cells in the lungs. Expression of CXCR6 on CD8 T cells was confirmed by flow cytometry. CONCLUSIONS: Our microarray analysis represents the first ex vivo study comparing gene expression profiles of CD8 T cells isolated from distinct sites after immunization with an adenoviral vector by different routes. It confirms earlier phenotypic data indicating that lung i.n. cells are more activated than lung i.d. CD8 T cells. The sustained expression of chemokines and activation genes enables CD8 T cells to remain in the lungs for extended periods after i.n. immunization. This may account for the early inhibition of M. tuberculosis growth observed in Ad85A i.n. immunized mice and explain the effectiveness of i.n. compared to parenteral immunization with this viral vector.

Nasal associated lymphoid tissue (NALT) contributes little to protection against aerosol challenge with Mycobacterium tuberculosis after immunisation with a recombinant adenoviral vaccine.

Intra-nasal administration of a recombinant adenovirus expressing Mycobacterium tuberculosis antigen 85A (Ad85A) has been shown to provide protection against challenge with M. tuberculosis. However the role of the upper respiratory tract associated lymphoid tissue, specifically the nasal associated lymphoid tissue (NALT), in providing protection has yet to be elucidated. Here we administered Ad85A to BALB/c mice alone or following BCG priming, using intranasal inocula targeting the whole respiratory tract or only the NALT, to show that Ad85A induces an immune response in the NALT insufficient to provide protection. Rather, Ad85A delivered through the respiratory tract must induce a deep lung immune response in order to protect against M. tuberculosis.

Immunization of mice with a recombinant adenovirus vaccine inhibits the early growth of Mycobacterium tuberculosis after infection.

BACKGROUND: In pulmonary Mycobacterium tuberculosis (Mtb) infection, immune responses are delayed compared to other respiratory infections, so that antigen-specific cells are not detected in the lungs earlier than day 14. Even after parenteral immunization with Bacille Calmette Guerin (BCG) or a subunit vaccine, the immune response after Mtb challenge is only slightly accelerated and the kinetics of pulmonary Mtb growth do not differ between naïve and immunized animals up to day 14. METHODS AND FINDINGS: Mice were immunized intranasally with a recombinant adenovirus expressing mycobacterial antigen 85A (Ad85A), challenged by aerosol with Mtb and the kinetics of Mtb growth in the lungs measured. Intranasal immunization with Ad85A inhibits Mtb growth in the early phase of infection, up to day 8. Protection is sustained for at least 7 months and correlates with the presence of antigen-specific activated effector CD8 T cells in the lungs. Antigen 85A-specific T cells respond to antigen presenting cells from the lungs of mice immunized with Ad85A 23 weeks previously, demonstrating the persistence of antigen in the lungs. CONCLUSIONS/SIGNIFICANCE: Intranasal immunization with Ad85A can inhibit early growth of Mtb because it establishes a lung antigen depot and maintains an activated lung-resident lymphocyte population. We propose that an optimal immunization strategy for tuberculosis should aim to induce both lung and systemic immunity, targeting the early and late phases of Mtb growth.

Toward the discovery of vaccine adjuvants: coupling in silico screening and in vitro analysis of antagonist binding to human and mouse CCR4 receptors.

BACKGROUND: Adjuvants enhance or modify an immune response that is made to an antigen. An antagonist of the chemokine CCR4 receptor can display adjuvant-like properties by diminishing the ability of CD4+CD25+ regulatory T cells (Tregs) to down-regulate immune responses. METHODOLOGY: Here, we have used protein modelling to create a plausible chemokine receptor model with the aim of using virtual screening to identify potential small molecule chemokine antagonists. A combination of homology modelling and molecular docking was used to create a model of the CCR4 receptor in order to investigate potential lead compounds that display antagonistic properties. Three-dimensional structure-based virtual screening of the CCR4 receptor identified 116 small molecules that were calculated to have a high affinity for the receptor; these were tested experimentally for CCR4 antagonism. Fifteen of these small molecules were shown to inhibit specifically CCR4-mediated cell migration, including that of CCR4(+) Tregs. SIGNIFICANCE: Our CCR4 antagonists act as adjuvants augmenting human T cell proliferation in an in vitro immune response model and compound SP50 increases T cell and antibody responses in vivo when combined with vaccine antigens of Mycobacterium tuberculosis and Plasmodium yoelii in mice.

Measurement of proliferation and disappearance of rapid turnover cell populations in human studies using deuterium-labeled glucose.

Cell proliferation may be measured in vivo by quantifying DNA synthesis with isotopically labeled deoxyribonucleotide precursors. Deuterium-labeled glucose is one such precursor which, because it achieves high levels of enrichment for a short period, is well suited to the study of rapidly dividing cells, in contrast to the longer term labeling achieved with heavy water ((2)H(2)O). As deuterium is non-radioactive and glucose can be readily administered, this approach is suitable for clinical studies. It has been widely applied to investigate human lymphocyte proliferation, but solid tissue samples may also be analyzed. Rate, duration and route (intravenous or oral) of [6,6-(2)H(2)]-glucose administration should be adapted to the target cell of interest. For lymphocytes, cell separation is best achieved by fluorescence activated cell sorting (FACS), although magnetic bead separation is an alternative. DNA is then extracted, hydrolyzed enzymatically and analyzed by gas chromatography mass spectrometry (GC/MS). Appropriate mathematical modeling is critical to interpretation. Typical time requirements are as follows: labeling, 10-24 h; sampling, approximately 3 weeks; DNA extraction/derivatization, 2-3 d; and GC/MS analysis, approximately 2 d.

Maintaining protection against invasive bacteria with protein-polysaccharide conjugate vaccines.

Polysaccharide-encapsulated organisms are the leading cause of bacterial meningitis and pneumonia in children. The use of protein-polysaccharide conjugate vaccines in developed countries over the past two decades has markedly decreased the burden of disease and mortality from these organisms through direct protection of the immunized and through herd immunity. In the next decade, the widespread use of conjugate vaccines in the developing world should prevent millions of deaths. In this Science and Society article, we describe how vaccine-induced immunity wanes rapidly after vaccination in early childhood and argue that strategies that sustain protection in the population must be considered.

Foot-and-mouth disease virus can induce a specific and rapid CD4+ T-cell-independent neutralizing and isotype class-switched antibody response in naïve cattle.

The role of T-lymphocyte subsets in recovery from foot-and-mouth disease virus (FMDV) infection in calves was investigated by administering subset-specific monoclonal antibodies. The depletion of circulating CD4(+) or WC1(+) gammadelta T cells was achieved for a period extending from before challenge to after resolution of viremia and peak clinical signs, whereas CD8(+) cell depletion was only partial. The depletion of CD4(+) cells was also confirmed by analysis of lymph node biopsy specimens 5 days postchallenge. Depletion with anti-WC1 and anti-CD8 antibodies had no effect on the kinetics of infection, clinical signs, and immune responses following FMDV infection. Three of the four CD4(+) T-cell-depleted calves failed to generate an antibody response to the nonstructural polyprotein 3ABC but generated a neutralizing antibody response similar to that in the controls, including rapid isotype switching to immunoglobulin G antibody. We conclude that antibody responses to sites on the surface of the virus capsid are T cell independent, whereas those directed against the nonstructural proteins are T cell dependent. CD4 depletion was found to substantially inhibit antibody responses to the G-H peptide loop VP1(135-156) on the viral capsid, indicating that responses to this particular site, which has a more mobile structure than other neutralizing sites on the virus capsid, are T cell dependent. The depletion of CD4(+) T cells had no adverse effect on the magnitude or duration of clinical signs or clearance of virus from the circulation. Overall, we conclude that CD4(+) T-cell-independent antibody responses play a major role in the resolution of foot-and-mouth disease in cattle.

Multifunctional, high-level cytokine-producing Th1 cells in the lung, but not spleen, correlate with protection against Mycobacterium tuberculosis aerosol challenge in mice.

Boosting bacillus Calmette-Guérin (BCG)-primed mice with a recombinant adenovirus expressing Mycobacterium tuberculosis Ag 85A by different administration routes has very different effects on protection against aerosol challenge with M. tuberculosis. Mice boosted intradermally make very strong splenic CD4 and CD8 Th1 cytokine responses to Ag 85A, but show no change in lung mycobacterial burden over BCG primed animals. In contrast, intranasally boosted mice show greatly reduced mycobacterial burden and make a much weaker splenic response but a very strong lung CD4 and CD8 response to Ag 85A and an increased response to purified protein derivative. This effect is associated with the presence in the lung of multifunctional T cells, with high median fluorescence intensity and integrated median fluorescence intensity for IFN-gamma, IL-2, and TNF. In contrast, mice immunized with BCG alone have few Ag-specific cells in the lung and a low proportion of multifunctional cells, although individual cells have high median fluorescence intensity. Successful immunization regimes appear to induce Ag-specific cells with abundant intracellular cytokine staining.

In silico identified CCR4 antagonists target regulatory T cells and exert adjuvant activity in vaccination.

Adjuvants are substances that enhance immune responses and thus improve the efficacy of vaccination. Few adjuvants are available for use in humans, and the one that is most commonly used (alum) often induces suboptimal immunity for protection against many pathogens. There is thus an obvious need to develop new and improved adjuvants. We have therefore taken an approach to adjuvant discovery that uses in silico modeling and structure-based drug-design. As proof-of-principle we chose to target the interaction of the chemokines CCL22 and CCL17 with their receptor CCR4. CCR4 was posited as an adjuvant target based on its expression on CD4(+)CD25(+) regulatory T cells (Tregs), which negatively regulate immune responses induced by dendritic cells (DC), whereas CCL17 and CCL22 are chemotactic agents produced by DC, which are crucial in promoting contact between DC and CCR4(+) T cells. Molecules identified by virtual screening and molecular docking as CCR4 antagonists were able to block CCL22- and CCL17-mediated recruitment of human Tregs and Th2 cells. Furthermore, CCR4 antagonists enhanced DC-mediated human CD4(+) T cell proliferation in an in vitro immune response model and amplified cellular and humoral immune responses in vivo in experimental models when injected in combination with either Modified Vaccinia Ankara expressing Ag85A from Mycobacterium tuberculosis (MVA85A) or recombinant hepatitis B virus surface antigen (rHBsAg) vaccines. The significant adjuvant activity observed provides good evidence supporting our hypothesis that CCR4 is a viable target for rational adjuvant design.

Primer: making sense of T-cell memory.

Protective memory is a key property of the immune system. Pathogen-associated molecular patterns of invading organisms deliver signals to pattern-recognition receptors that activate the innate immune system. Ligation of the T-cell receptor by peptides bound to MHC antigens and presented by dendritic cells, together with signals produced by the activated innate immune system, initiate T-cell responses. The nature of the T-cell response, consisting of phases of clonal expansion and contraction, and differentiation to effector and memory cells, however, is determined both by the properties of the antigen and the co-stimuli produced by the innate immune system. Short-lived effector and longer-lived memory T cells are generated during primary responses; after the death of most of the effectors, memory cells remain. Memory cells are heterogeneous in phenotype and function; subsets include the relatively quiescent central and more activated effector memory cells, as well as cells able to promote inflammation, help antibody production or regulate other immune responses. Understanding the properties of memory cells will help in the rational design of vaccines for 'difficult' organisms or cancer, as well as immunotherapies for autoimmune diseases.

Serotype-specific and age-dependent generation of pneumococcal polysaccharide-specific memory B-cell and antibody responses to immunization with a pneumococcal conjugate vaccine.

Glycoconjugate vaccines have dramatically reduced the incidence of encapsulated bacterial diseases in toddlers under 2 years of age, but vaccine-induced antibody levels in this age group wane rapidly. We immunized adults and 12-month-old toddlers with heptavalent pneumococcal conjugate vaccine to determine differences in B-cell and antibody responses. The adults and 12-month-old toddlers received a pneumococcal conjugate vaccine. The toddlers received a second dose at 14 months of age. The frequencies of diphtheria toxoid and serotype 4, 14, and 23F polysaccharide-specific plasma cells and memory B cells were determined by enzyme-linked immunospot assay. The toddlers had no preexisting polysaccharide-specific memory B cells or serum immunoglobulin G (IgG) antibody but had good diphtheria toxoid-specific memory responses. The frequencies of plasma cells and memory B cells increased by day 7 (P < 0.0001) in the adults and the toddlers following a single dose of conjugate, but the polysaccharide responses were significantly lower in the toddlers than in the adults (P = 0.009 to <0.001). IgM dominated the toddler antibody responses, and class switching to the IgG was serotype dependent. A second dose of vaccine enhanced the antibody and memory B-cell responses in the toddlers but not the ex vivo plasma cell responses. Two doses of pneumococcal conjugate vaccine are required in toddlers to generate memory B-cell frequencies and antibody class switching for each pneumococcal polysaccharide equivalent to that seen in adults.