Regulation of Th-1 T cell-dominated immunity to Neisseria meningitidis within the human mucosa

Neisseria meningitidis is commonly carried asymptomatically in the upper respiratory tract and only occasionally invades the bloodstream and meninges to cause disease. Naturally acquired immunity appears protective but the nature of the cellular immune response within the mucosa is uncertain. We show that following in vitro stimulation with N. meningitidis serogroup B (MenB) antigens, approximately 66% of the dividing mucosal CD4(+)CD45RO(+) memory population express the Th1-associated IL18-R while the remainder express CRTH2, a Th2-associated marker. The pro-inflammatory bias of this anti-MenB response is not evident in blood, demonstrating compartmentalization at the induction site; and occurs in the presence or absence of lipopolysacharide indicating that these responses are already fully committed. Depletion of CD25(+) cells reveals suppression of the effector CD4(+) T cell response restricted to the mucosa and most marked in children (i.e. those at greatest risk of disease). Mucosal T-regulatory cell (Treg) activity is partially overcome by blocking the human glucocorticoid-induced TNF receptor (GITR) and is not seen following stimulation with antigens from another mucosal pathogen, influenza virus. Pro-inflammatory, antimeningococcal T cell responses may limit invasive disease at the mucosa but Treg induction while reducing immunopathological damage, may also restrict the effectiveness of the protective response, particularly in children.     

Induction in humans of CD8+ and CD4+ T cell and antibody responses by sequential immunization with malaria DNA and recombinant protein

Vaccine-induced protection against diseases like malaria, AIDS, and cancer may require induction of Ag-specific CD8(+) and CD4(+) T cell and Ab responses in the same individual. In humans, a recombinant Plasmodium falciparum circumsporozoite protein (PfCSP) candidate vaccine, RTS,S/adjuvant system number 2A (AS02A), induces T cells and Abs, but no measurable CD8(+) T cells by CTL or short-term (ex vivo) IFN-gamma ELISPOT assays, and partial short-term protection. P. falciparum DNA vaccines elicit CD8(+) T cells by these assays, but no protection. We report that sequential immunization with a PfCSP DNA vaccine and RTS,S/AS02A induced PfCSP-specific Abs and Th1 CD4(+) T cells, and CD8(+) cytotoxic and Tc1 T cells. Depending upon the immunization regime, CD4(+) T cells were involved in both the induction and production phases of PfCSP-specific IFN-gamma responses, whereas, CD8(+) T cells were involved only in the production phase. IFN-gamma mRNA up-regulation was detected in both CD45RA(-) (CD45RO(+)) and CD45RA(+)CD4(+) and CD8(+) T cell populations after stimulation with PfCSP peptides. This finding suggests CD45RA(+) cells function as effector T cells. The induction in humans of the three primary Ag-specific adaptive immune responses establishes a strategy for developing immunization regimens against diseases in desperate need of vaccines.     

Analysis of Neisseria lactamica antigens putatively implicated in acquisition of natural immunity to Neisseria meningitidis

Sera from healthy human volunteers, patients convalescent from meningococcal meningitis, and mice immunized with outer membrane proteins from Neisseria meningitidis and Neisseria lactamica strains were used to analyze and identify antigens cross-reactive to both neisserial species. All classes of meningococcal proteins except class 1 (PorA) and class 5 cross-reacted with N. lactamica proteins and two other proteins of 65 and 55 kDa (an iron-regulated protein). Results obtained with the mouse sera demonstrate that cross-reactive antibodies can be elicited by either N. meningitidis or N. lactamica. These results support the suggestion that N. lactamica contributes to the development of natural immunity against N. meningitidis during the first years of life. The use of vaccines containing proteins other than PorA could interfere in colonization of mucosal surfaces by N. lactamica, hampering the natural mechanisms of immunity acquisition in humans. Only convalescent sera reacted with the 55 and 65 kDa proteins, which suggests that they might be relevant for pathogenicity.     

Spermine, a natural polyamine, suppresses LFA-1 expression on human lymphocyte

Natural polyamines, spermine, spermidine, and putrescine, play a pivotal role in the regulation of gene expression; therefore, the age-dependent decreases and the disease-dependent increases in polyamine synthesis suggest a possible contribution of polyamines to the age-related and disease-associated changes in cellular function. In this study, we examined the effects of polyamines on the cellular function and the expression of adhesion molecules on human PBMCs from healthy volunteers. Flow cytometry revealed that PBMCs cultured with spermine decreased mean fluorescent intensities (MFIs) of CD11a and CD18 in the lymphocyte light-scattered region, but not in the monocyte region. This suppression was observed in a dose- and time-dependent manner and found nonspecifically on all cell subsets we tested (CD3(+), CD4(+), CD8(+), CD19(+), CD45RA(+), CD45RO(+), CD4(+)CD45RA(+), CD4(+)CD45RO(+), CD8(+)CD45RA(+), CD8(+)CD45RO(+)). The decreases of CD11a and CD18 MFIs were accompanied by the decrease in adherent capacity of PBMCs to HUVECs. Spermine did not hinder cell activities or cell viability. Among 42 healthy volunteers (mean, 49.5 years old; from 26 to 69), blood spermine levels inversely correlated with the CD11a MFIs of cells in the lymphocyte region (r = -0.48; p = 0.001), but not with those in the monocyte region. The effects of spermidine seemed weaker than those of spermine, and blood spermidine levels had no correlation with CD11a MFIs of the lymphocyte region. Putrescine had no effect on the expressions of membrane molecules. Polyamines, especially spermine, decrease LFA-1 (CD11a/CD18) expression on human lymphocyte and adhesion capacity of PBMCs to HUVECs.     

Genetic and immunologic characterization of a novel serotype 4, 15 strain of Neisseria meningitidis

The porin proteins of Neisseria meningitidis are important components of outer membrane protein (OMP) vaccines. The class 3 porin gene, porB, of a novel serogroup B, serotype 4, 15 isolate from Chile (Ch501) was found to be VR1-4, VR2-15, VR3-15 and VR4-15 by porB variable region (VR) typing. Rabbit immunization studies using outer membrane vesicles revealed immunodominance of individual PorB (class 3) VR epitopes. The predominant anti-Ch501 PorB response was directed to the VR1 epitope. Anti-PorB VR1 mediated killing was suggested by the bactericidal activity of Ch501 anti-sera against a type 4 strain not expressing PorA or class 5 OMPs. Studies that examine the molecular epidemiology of individual porB VRs, and the immune responses to PorB epitopes, may contribute to the development of broadly protective group B meningococcal vaccines.     

Accessory cell function of airway epithelial cells

Accessory cell function of airway epithelial cells. We previously demonstrated that airway epithelial cells (AECs) have many features of accessory cells, including expression of class II molecules CD80 and CD86 and functional Fcgamma receptors. We have extended these studies to show that freshly isolated AECs have mRNA for cathepsins S, V, and H [proteases important in antigen (Ag) presentation], invariant chain, human leukocyte antigen (HLA)-DM-alpha and HLA-DM-beta, and CLIP, an invariant chain breakdown product. A physiologically relevant Ag, ragweed, was colocalized with HLA-DR in AECs, and its uptake was increased by granulocyte-macrophage colony-stimulating factor and IFN-gamma treatments, which had no effect on CD80 and CD86 expression. We demonstrate the presence of other costimulatory molecules, including B7h and B7-H1, on AECs and the increased expression of B7-H1 on AECs after treatment with granulocyte-macrophage colony-stimulating factor and IFN-gamma. Finally, we compared T cell proliferation after allostimulation with AECs and dendritic cells (DCs). The precursor frequency of peripheral blood T cells responding to AECs was 0.264% compared with 0.55% for DCs. DCs stimulated CD45RO(+), CD45RA(+), CCR7(+) and CCR7(-)CD4(+), and CD8(+) T cells, whereas AECs stimulated only CD45RO(+), CD45RA(-), CCR7(-), CD4(+), and CD8(+) T cells. There was no difference in cytokine production, type of memory T cells stimulated (effector vs. long-term memory), or apoptosis by T cells cocultured with AECs and DCs. The localization of AECs exposed to the external environment may make them important in the regulation of local immune responses.     

Anti-PorA antibodies elicited by immunization with peptides conjugated to P64k

To increase the humoral immune response against two cyclic synthetic peptides, derived from variable regions within the outer membrane meningococcal protein PorA (subtypes 19 and 15), we conjugated the peptides to P64k, a novel carrier protein from the same bacterium expressed in Escherichia coli. In addition, one of these peptides was restricted to a linear conformation before it was chemically coupled to the carrier. The conjugates were administered to mice in a three-dose immunization schedule, resulting in a potent anti-peptide immune response, which suggested that chemical conjugation to this carrier provided T-cell help. Antisera directed to the three conjugates reacted with Neisseria meningitidis outer membrane PorA upon immunoblot analysis. Moreover, in two out of three conjugates, the anti-peptide sera reacted with native meningococcal outer membrane vesicles in ELISA.     

Crystal structure of an Anti-meningococcal subtype P1.4 PorA antibody provides basis for peptide-vaccine design

In various western countries, subtype P1.4 of Neisseria meningitidis serogroup B causes the greatest incidence of meningococcal disease. To investigate the molecular recognition of this subtype, we crystallised a peptide (P1HVVVNNKVATH(P11)), corresponding to the subtype P1.4 epitope sequence of outer membrane protein PorA, in complex with a Fab fragment of the bactericidal antibody MN20B9.34 directed against this epitope. Structure determination at 1.95 A resolution revealed a unique complex of one P1.4 antigen peptide bound to two identical Fab fragments. One Fab recognises the putative epitope residues in a 2:2 type I beta-turn at residues P5NNKV(P8), whereas the other Fab binds the C-terminal residues of the peptide that we consider a crystallisation artefact. Interestingly, recognition of the P1.4 epitope peptide is mediated almost exclusively through the complementarity-determining regions of the heavy chain. We exploited the observed turn conformation for designing conformationally restricted cyclic peptides for use as a peptide vaccine. The conformational stability of the two peptide designs was assessed by molecular dynamics simulations. Unlike the linear peptide, both cyclic peptides, conjugated to tetanus toxoid as a carrier protein, elicited antibody responses in mice that recognised meningococci of subtype P1.7-2,4. Serum bactericidal assays showed that some, but not all, of the sera induced with the cyclic peptide conjugates could activate the complement system with titres that were very high compared to the titres induced by complete PorA protein in its native conformation administered in outer membrane vesicles.     

Regulation of trafficking receptor expression in human forkhead box P3+ regulatory T cells

Forkhead Box P3(+) (FOXP3(+)) T cells are regulatory cells important for maintaining immune tolerance. While chemokine- and other homing-receptors are important for T cell migration, it has been unclear how they are regulated in FOXP3(+) T cells. We thoroughly investigated, ex vivo and in vitro, the regulation of chemokine receptor expression on human FOXP3(+) T cells in neonatal cord blood, adult peripheral blood, and tonsils. We found that human FOXP3(+) T cells undergo changes in trafficking receptors according to their stages of activation and differentiation. FOXP3(+) T cells are divided into CD45RA(+) (naive type) and CD45RO(+) (memory type) FOXP3(+) T cells in neonatal blood, adult blood, and tonsils. CD45RA(+)FOXP3(+) T cells mainly express lymphoid tissue homing receptors (CD62L, CCR7, and CXCR4), while CD45RO(+)FOXP3(+) T cells highly express both Th1 and Th2-associated trafficking receptors along with the lymphoid tissue homing receptors at reduced frequencies. Up-regulation of Th1/Th2-associated trafficking receptors begins with activation of CD45RA(+)FOXP3(+) T cells and is completed after their differentiation to CD45RO(+) cells. Some chemokine receptors such as CXCR5 and CXCR6 are preferentially expressed by many FOXP3(+) cells at a specific stage (CD69(+)CD45RO(+)) in tonsils. Our in vitro differentiation study demonstrated that CD45RA(+)FOXP3(+) T cells indeed undergo chemokine receptor switch from CD45RA(+) (secondary lymphoid tissue homing) to CD45RO(+) type (lymphoid and nonlymphoid tissue homing). The orderly regulation of trafficking receptors in FOXP3(+) T cells according to stages of differentiation and activation is potentially important for their tissue-specific migration and regulation of immune responses in humans.     

A human memory T cell subset with stem cell-like properties

Immunological memory is thought to depend on a stem cell-like, self-renewing population of lymphocytes capable of differentiating into effector cells in response to antigen re-exposure. Here we describe a long-lived human memory T cell population that has an enhanced capacity for self-renewal and a multipotent ability to derive central memory, effector memory and effector T cells. These cells, specific to multiple viral and self-tumor antigens, were found within a CD45RO(-), CCR7(+), CD45RA(+), CD62L(+), CD27(+), CD28(+) and IL-7Rα(+) T cell compartment characteristic of naive T cells. However, they expressed large amounts of CD95, IL-2Rβ, CXCR3, and LFA-1, and showed numerous functional attributes distinctive of memory cells. Compared with known memory populations, these lymphocytes had increased proliferative capacity and more efficiently reconstituted immunodeficient hosts, and they mediated superior antitumor responses in a humanized mouse model. The identification of a human stem cell-like memory T cell population is of direct relevance to the design of vaccines and T cell therapies.     

Adjuvant Effects Elicited by Novel Oligosaccharide Variants of Detoxified Meningococcal Lipopolysaccharides on Neisseria meningitidis Recombinant PorA Protein: A Comparison in Mice

Neisseria meningitidis lipopolysaccharide (LPS) has adjuvant properties that can be exploited to assist vaccine immunogenicity. The modified penta-acylated LPS retains the adjuvant properties of hexa-acylated LPS but has a reduced toxicity profile. In this study we investigated whether two modified glycoform structures (LgtE and IcsB) of detoxified penta-acylated LPS exhibited differential adjuvant properties when formulated as native outer membrane vesicles (nOMVs) as compared to the previously described LgtB variant. Detoxified penta-acylated LPS was obtained by disruption of the lpxL1 gene (LpxL1 LPS), and three different glycoforms were obtained by disruption of the lgtB, lgtE or icsB genes respectively. Mice (mus musculus) were immunized with a recombinant PorA P1.7-2,4 (rPorA) protein co-administered with different nOMVs (containing a different PorA serosubtype P1.7,16), each of which expressed one of the three penta-acylated LPS glycoforms. All nOMVs induced IgG responses against the rPorA, but the nOMVs containing the penta-acylated LgtB-LpxL1 LPS glycoform induced significantly greater bactericidal activity compared to the other nOMVs or when the adjuvant was Alhydrogel. Compared to LgtE or IcsB LPS glycoforms, these data support the use of nOMVs containing detoxified, modified LgtB-LpxL1 LPS as a potential adjuvant for future meningococcal protein vaccines.     

Protective mucosal immunity in aging is associated with functional CD4+ T cells in nasopharyngeal-associated lymphoreticular tissue

Our previous studies showed that mucosal immunity was impaired in 1-year-old mice that had been orally immunized with OVA and native cholera toxin (nCT) as mucosal adjuvant. In this study, we queried whether similar immune dysregulation was also present in mucosal compartments of mice immunized by the nasal route. Both 1-year-old and young adult mice were immunized weekly with three nasal doses of OVA and nCT or with a nontoxic chimeric enterotoxin (mutant cholera toxin-A E112K/B subunit of native labile toxin) from Brevibacillus choshinensis. Elevated levels of OVA-specific IgG Abs in plasma and secretory IgA Abs in mucosal secretions (nasal washes, saliva, and fecal extracts) were noted in both young adult and 1-year-old mice given nCT or chimeric enterotoxin as mucosal adjuvants. Significant levels of OVA-specific CD4(+) T cell proliferative and OVA-induced Th1- and Th2-type cytokine responses were noted in cervical lymph nodes and spleen of 1-year-old mice. In this regard, CD4(+), CD45RB(+) T cells were detected in greater numbers in the nasopharyngeal-associated lymphoreticular tissues of 1-year-old mice than of young adult mice, but the same did not hold true for Peyer's patches or spleen. One-year-old mice given nasal tetanus toxoid plus the chimeric toxin as adjuvant were protected from lethal challenge with tetanus toxin. This result reinforced our findings that age-associated immune alterations occur first in gut-associated lymphoreticular tissues, and thus nasal delivery of vaccines for nasopharyngeal-associated lymphoreticular tissue-based mucosal immunity offers an attractive possibility to protect the elderly.     

Memory T cells constitute a subset of the human CD8+CD45RA+ pool with distinct phenotypic and migratory characteristics.

Using HLA class I-viral epitope tetramers to monitor herpes virus-specific CD8(+) T cell responses in humans, we have shown that a significant fraction of responding cells revert from a CD45RO(+) to a CD45RA(+) state after priming. All tetramer-binding CD45RA(+) cells, regardless of epitope specificity, expressed a phenotype LFA-1(high)CCR7(low) that was stable for at least 10 years in infectious mononucleosis patients and indefinitely in asymptomatic carriers. CD8(+)CD45RA(+)LFA-1(high) cells were not present in cord blood but in adults account for up to 50% of CD8(+)CD45RA(+) cells. These CD45RA(+)LFA-1(high) cells have significantly shorter telomeres than CD45RA(+)LFA-1(low) cells, suggesting that the latter represent a naive population, while the former are memory cells. CD45RA(+) memory cells are a stable population of noncycling cells, but on stimulation they are potent producers of IFN-gamma, while naive CD8(+) cells produce only IL-2. The chemokine receptor profile and migratory potential of CD45RA(+) memory cells is very similar to CD45RO(+) cells but different to naive CD8 cells. In accord with this, CD45RA(+) memory cells were significantly underrepresented in lymph nodes, but account for virtually all CD8(+)CD45RA(+) T cells in peripheral tissues of the same individuals.     

An OMV Vaccine Derived from a Capsular Group B Meningococcus with Constitutive FetA Expression: Preclinical Evaluation of Immunogenicity and Toxicity

Following the introduction of effective protein-polysaccharide conjugate vaccines against capsular group C meningococcal disease in Europe, meningococci of capsular group B remain a major cause of death and can result in debilitating sequelae. The outer membrane proteins PorA and FetA have previously been shown to induce bactericidal antibodies in humans. Despite considerable antigenic variation among PorA and FetA OMPs in meningococci, systematic molecular epidemiological studies revealed this variation is highly structured so that a limited repertoire of antigenic types is congruent with the hyperinvasive meningococcal lineages that have caused most of the meningococcal disease in Europe in recent decades. Here we describe the development of a prototype vaccine against capsular group B meningococcal infection based on a N. meningitidis isolate genetically engineered to have constitutive expression of the outer membrane protein FetA. Deoxycholate outer membrane vesicles (dOMVs) extracted from cells cultivated in modified Frantz medium contained 21.8% PorA protein, 7.7% FetA protein and 0.03 μg LPS per μg protein (3%). The antibody response to the vaccine was tested in three mouse strains and the toxicological profile of the vaccine was tested in New Zealand white rabbits. Administration of the vaccine, MenPF-1, when given by intramuscular injection on 4 occasions over a 9 week period, was well tolerated in rabbits up to 50 μg/dose, with no evidence of systemic toxicity. These data indicated that the MenPF-1 vaccine had a toxicological profile suitable for testing in a phase I clinical trial.     

A novel mimetic antigen eliciting protective antibody to Neisseria meningitidis.

Molecular mimetic Ags are of considerable interest as vaccine candidates. Yet there are few examples of mimetic Ags that elicit protective Ab against a pathogen, and the functional activity of anti-mimetic Abs has not been studied in detail. As part of the Neisseria meningitidis serogroup B genome sequencing project, a large number of novel proteins were identified. Herein, we provide evidence that genome-derived Ag 33 (GNA33), a lipoprotein with homology to Escherichia coli murein transglycosylase, elicits protective Ab to meningococci as a result of mimicking an epitope on loop 4 of porin A (PorA) in strains with serosubtype P1.2. Epitope mapping of a bactericidal anti-GNA33 mAb using overlapping peptides shows that the mAb recognizes peptides from GNA33 and PorA that share a QTP sequence that is necessary but not sufficient for binding. By flow cytometry, mouse antisera prepared against rGNA33 and the anti-GNA33 mAb bind as well as an anti-PorA P1.2 mAb to the surface of eight of nine N. meningitidis serogroup B strains tested with the P1.2 serosubtype. Anti-GNA33 Abs also are bactericidal for most P1.2 strains and, for susceptible strains, the activity of an anti-GNA33 mAb is similar to that of an anticapsular mAb but less active than an anti-P1.2 mAb. Anti-GNA Abs also confer passive protection against bacteremia in infant rats challenged with P1.2 strains. Thus, GNA33 represents one of the most effective immunogenic mimetics yet described. These results demonstrate that molecular mimetics have potential as meningococcal vaccine candidates.     

FetA Antibodies Induced by an Outer Membrane Vesicle Vaccine Derived from a Serogroup B Meningococcal Isolate with Constitutive FetA Expression

Invasive meningococcal disease causes over 3500 cases each year in Europe, with particularly high incidence among young children. Among serogroup B meningococci, which cause most of the cases, high diversity in the outer membrane proteins (OMPs) is observed in endemic situations; however, comprehensive molecular epidemiological data are available for the diversity and distribution of the OMPs PorA and FetA and these can be used to rationally design a vaccine with high coverage of the case isolates. The aim of this study was to determine whether outer membrane vesicles (OMVs) derived from an isolate with constitutive FetA expression (MenPF-1 vaccine) could be used to induce antibodies against both the PorA and FetA antigens. The immunogenicity of various dose levels and number of doses was evaluated in mice and rabbits, and IgG antibody responses tested against OMVs and recombinant PorA and FetA proteins. A panel of four isogenic mutants was generated and used to evaluate the relative ability of the vaccine to induce serum bactericidal activity (SBA) against FetA and PorA. Sera from mice were tested in SBA against the four target strains. Results demonstrated that the MenPF-1 OMVs were immunogenic against PorA and FetA in both animal models. Furthermore, the murine antibodies induced were bactericidal against isogenic mutant strains, suggesting that antibodies to both PorA and FetA were functional. The data presented indicate that the MenPF-1 vaccine is a suitable formulation for presenting PorA and FetA OMPs in order to induce bactericidal antibodies, and that proceeding to a Phase I clinical trial with this vaccine candidate is justified.     

Identification of naive or antigen-experienced human CD8(+) T cells by expression of costimulation and chemokine receptors: analysis of the human cytomegalovirus-specific CD8(+) T cell response

Human CMV (HCMV) infection provides an informative model of how long term human CD8(+) T cell memory is maintained in the presence of Ag. To clarify the phenotypic identity of Ag-experienced human CD8(+) T cells in vivo, we determined the expression of costimulation and chemokine receptors on Ag-specific CD8(+) T cells by quantifying individual virus-specific clones in different cell populations using TCR clonotypic probing. In healthy HCMV carriers, expanded CD8(+) clones specific for either HCMV tegument protein pp65 or immediate-early protein IE72 are found in both CD45RO(high) cells and the subpopulation of CD45RA(high) cells that lack the costimulatory molecule CD28. In contrast to previous suggested models of CD8(+) T cell memory, we found that in healthy virus carriers highly purified CD28(-)CD45RA(high)CCR7(-) cells are not terminally differentiated, because following stimulation in vitro with specific HCMV peptide these cells underwent sustained clonal proliferation, up-regulated CD45RO and CCR5, and showed strong peptide-specific cytotoxic activity. In an individual with acute primary HCMV infection, HCMV pp65-specific CD8(+) T cells are predominantly CD28(-)CD45RO(high)CCR7(-). During convalescence, an increasing proportion of pp65-specific CD8(+) T cells were CD28(-)CD45RA(high)CCR7(-). We conclude that naive human CD8(+) T cells are CD28(+)CD45RA(high), express CCR7 but not CCR6, and are predominantly CD27(+) and L-selectin CD62 ligand-positive. The phenotype CD27(+)CD45RA(high) should not be used to identify naive human CD8(+) T cells, because CD27(+)CD45RA(high) cells also contain a significant subpopulation of CD28(-)CD27(+) Ag-experienced expanded clones. Thus CD8(+) T cell memory to HCMV is maintained by cells of expanded HCMV-specific clones that show heterogeneity of activation state and costimulation molecular expression within both CD45RO(high) and CD28(-)CD45RA(high) T cell pools.     

Differentiation of human CD8(+) T cells from a memory to memory/effector phenotype

Previous studies of perforin expression and cytokine production in subsets of peripheral human CD45RA(-)CD8(+) T cells with different CD28/CD27 phenotypes showed that CD28(+)CD45RA(-)CD8(+) and CD27(+)CD45RA(-)CD8(+) T cells have characteristics of memory T cells, whereas CD28(-)CD45RA(-)CD8(+) and CD27(-)CD45RA(-)CD8(+) T cells have characteristics of both memory and effector T cells. However, the differentiation pathway from memory CD8(+) T cells into memory/effector CD8(+) T cells has not been completely clarified. We investigated this differentiation pathway using EBV- and human CMV (HCMV)-specific CD8(+) T cells. Three subsets of CD45RA(-)CD8(+) T cells were observed in both total CD8(+) T cells and EBV- or HCMV-specific CD8(+) T cells: CD27(+)CD28(+), CD27(+)CD28(-), and CD27(-)CD28(-). A significant number of the CD27(-)CD28(+) subset was observed in total CD8 T cells. However, this subset was barely detectable in EBV- or HCMV-specific CD8(+) T cells. Analysis of perforin expression and cytotoxic activity in the first three subsets suggested the following differentiation pathway: CD27(+)CD28(+)CD45RA(-)-->CD27(+)CD28(-)CD45RA(-)-->CD27(-)CD28(-)CD45RA(-). This was supported by the observation that the frequency of CCR5(+) cells and CCR7(+) cells decreased during this sequence. Analysis of CCR5 and CCR7 expression in the CD27(+)CD28(+) memory cell subset demonstrated the presence of three CCR5/CCR7 populations: CCR5(-)CCR7(+), CCR5(+)CCR7(+), and CCR5(+)CCR7(-). These findings suggested the following differentiation pathway: CD27(+)CD28(+)CD45RA(-) (CCR5(-)CCR7(+)-->CCR5(+)CCR7(+)-->CCR5(+)CCR7(-))-->CD27(+)CD28(-)CD45RA(-)-->CD27(-)CD28(-)CD45RA(-). The presence of a CD27(-)CD28(+) subset with a CCR5(+)CCR7(-) phenotype implies a specialized role for this subset in the differentiation of CD8(+) T cells.     

CCL5 modulates pneumococcal immunity and carriage

Understanding the requirements for protection against pneumococcal carriage and pneumonia will greatly benefit efforts in controlling these diseases. Recently, it has been shown that genetic polymorphisms can result in diminished expression of CCL5, which results in increased susceptibility to and progression of infectious diseases. We show that CCL5, together with Th cytokine mRNA expression, is temporally up-regulated during pneumococcal carriage. To determine the contribution of CCL5 to pneumococcal surface antigen A-specific humoral and cellular pneumococcal immunity, mice were treated with anti-CCL5 or control Abs before and during Streptococcus pneumoniae strain EF3030-challenge for the initiation of carriage. CCL5 blockade resulted in a decrease of CD4(+) and CD8(+) T cells as well as CD11b(+) cells in the spleen, cervical lymph node, lung, and nasopharyngeal associated lymphoid tissue during the recognition phase of the pneumococcal adaptive immune response. CCL5 blockade significantly reduced the Ag-specific IgG2a and IgG1 Abs in serum and IgA Ab levels in nasal washes. These decreases also corresponded to reductions in Ag-specific T cell (mucosal and systemic) responses. CCL5 inhibition resulted in decreasing the quantity of IL-4- and IFN-gamma-secreting CD4(+) T cells and increasing the number of Ag-specific IL-10-producing CD4(+) T cells; these changes combined also corresponded with the transition from pneumococcal carriage to lethal pneumonia. These data suggest that CCL5 is an essential factor for the induction and maintenance of protective pneumococcal immunity.     

Immunogenicity of in vitro folded outer membrane protein PorA of Neisseria meningitidis

In vitro folded and the denatured form of PorA P1.6 from Neisseria meningitidis strain M990 were used for immunization studies in mice. Previously, the antigen was isolated from cytoplasmic inclusion bodies, folded and purified. Its immunogenicity without adjuvant appeared to be low. The addition of the adjuvant QuilA, but not of galE lipooligosaccharide, considerably enhanced the immunogenicity. Moreover, when immunized with folded PorA P1.6 plus QuilA, a clear switch towards the IgG2a subclass of antibodies and concomitantly, the appearance of serum bactericidal activity, which is believed to be important for protective immunity, was observed. Hence, a tool for preparing vaccines against serogroup B meningococci devoid of endotoxin is available.