André F. M. Verheul

Synthetic 6B Di-, Tri-, and Tetrasaccharide-Protein Conjugates Contain Pneumococcal Type 6A and 6B Common and 6B- Specific Epitopes That Elicit Protective Antibodies in Mice

ABSTRACT The immunogenicity and protective capacity of Streptococcus pneumoniae 6B capsular polysaccharide (PS)-derived synthetic phosphate-containing disaccharide (Rha-ribitol-P-), trisaccharide (ribitol-P-Gal-Glc-), and tetrasaccharide (Rha-ribitol-P-Gal-Glc-)-protein conjugates in rabbits and mice were studied. In rabbits, all saccharides conjugated to keyhole limpet hemocyanin (KLH) evoked high levels of pneumococcal (Pn) type 6B antibodies that facilitated type-specific phagocytosis. Unlike the disaccharide rabbit antisera, tri- and tetrasaccharide rabbit antisera also reacted with 6A PS in an enzyme-linked immunosorbent assay (ELISA) and promoted phagocytosis of 6A pneumococci. All these rabbit antisera passively protected mice against a Pn 6B challenge. The disaccharide conjugate-induced antiserum, however, failed to protect mice against a 6A challenge. In mice, phagocytic and protective anti-Pn 6B antibodies were only induced by the tetrasaccharide conjugate and not by PS 6B or PS 6B-protein conjugates. These antibodies did not cross-react with 6A PS in ELISA and were unable to phagocytize 6A pneumococci. In conclusion, the disaccharide and tetrasaccharide conjugates already contain epitopes capable of inducing 6B-specific, fully protective antibodies in rabbits and mice, respectively.

Cytokine Profiles in Cerebrospinal Fluid of Human Immunodeficiency Virus—Infected Patients with Cryptococcal Meningitis: No Leukocytosis despite High Interleukin-8 Levels

Cytokine levels were studied in the cerebrospinal fluid (CSF) of 16 adults with cryptococcal meningitis (CM). Low levels of tumor necrosis factor (TNF)-alpha and interferon-gamma, high levels of interleukin (IL)-1beta, IL-6, and IL-8, and the presence of IL-10 were documented. There were no significant differences in levels of TNF-alpha and interferon-gamma for CM and control patients. Mean CSF levels of IL-1beta (139.5 pg/mL), IL-6 (346 pg/mL), IL-8 (1160 pg/mL), and IL-10 (9.27 pg/mL) were significantly (P < .01) elevated in CM patients compared with levels in control patients. Despite the high CSF levels of IL-8, minimal leukocytosis was seen. Significant correlations between cryptococcal antigen titers and IL-10 levels (r = .8, P < .05), protein and cryptococcal antigen titer (r = .9, P < .05), and protein and IL-10 levels (r = .8, P < .05) were found.

Induction of Cell-Mediated Immunity against Mycobacterium tuberculosis Using DNA Vaccines Encoding Cytotoxic and Helper T-Cell Epitopes of the 38-Kilodalton Protein

ABSTRACT Cell-mediated immune responses are crucial in the protection against tuberculosis. In this study, we constructed DNA vaccines encoding cytotoxic T lymphocytes (CTL) and T helper cell (Th) epitopes of the 38-kDa lipoglycoprotein of Mycobacterium tuberculosis and analyzed and compared their immunogenicities with that of pXJ38, a DNA vaccine encoding the entire 38-kDa protein (X. Zhu, N. Venkataprasad, H. S. Thangaraj, M. Hill, M. Singh, J. Ivanyi, and H. M. Vordermeier, J. Immunol. 158:5921–5926, 1997). Plasmid DNAs encoding a CTL epitope, P3 (pP3), a Th epitope (vTh), or both the Th and the P3 epitopes (pThP3) were prepared and tested in C57BL6/J (H-2b) mice. Our results confirmed that DNA immunization with pXJ38 induces strong CD8+CTL and Th1 responses (high gamma interferon [IFN-γ], low interleukin-4 [IL-4]). Coadministration of plasmid DNAs encoding a Th epitope with those encoding a CTL epitope (vTh+pP3) elicited both antigen-specific CD8+ CTL and Th1 responses. High levels of IFN-γ were secreted by spleen cells from all plasmid DNA-vaccinated mice after in vitro stimulation with the recombinant 38-kDa protein. Small or undetectable amounts of IL-4 were observed, which indicates the induction of a Th1-like response. Multiple-epitope vaccination by vTh+pP3 or pThP3 resulted in a broader Th1 response to peptide or epitopes than the single-epitope plasmid DNAs. Antigen-specific immunoglobulin G2a was only detected in sera from mice immunized with the plasmid pXJ38, and not in mice immunized with the epitope-based DNA vaccines. Thus, the absence of an antibody response after immunization with epitope plasmid DNAs and their ability to trigger only a specific cellular immune response may prove to be important advantages for a vaccine against tuberculosis.

Are the Opsonophagocytic Activities of Antibodies in Infant Sera Measured by Different Pneumococcal Phagocytosis Assays Comparable

ABSTRACT Host protection against Streptococcus pneumoniae is mainly mediated by opsonin-dependent phagocytosis. Several techniques for measuring opsonophagocytic activity (OPA) of antibodies to S. pneumoniae have been standardized and used. These include the viable cell-assay, flow-cytometric assays, and an assay utilizing radiolabeled bacteria. Using these different methods, we measured the OPA of antibodies to S. pneumoniae types 6B and 19F from the sera of infants immunized with a pneumococcal conjugate vaccine, PncCRM. Generally, the results obtained by the various techniques correlated well, although serotype-specific differences were found (6B,r = 0.78 to 0.95, P < 0.001; 19F,r = 0.50 to 0.84, P < 0.001). The same serotype-specific differences were observed for the relationship between the concentrations of specific immunoglobulin G antibodies measured by enzyme immunoassay and the OPA. Since the sensitivities of the OPA assays differed, the most prominent discrepancies between the techniques were found at low antibody concentrations.

Fcγ Receptor Polymorphisms Determine the Magnitude of In Vitro Phagocytosis of Streptococcus pneumoniae Mediated by Pneumococcal Conjugate Sera

Fcgamma receptors show two genetically determined polymorphisms: the biallelic FcgammaRIIa-R131 and -H131 polymorphism and the NA1/NA2 FcgammaIIIb polymorphism. Using 10 pre- and postconjugate vaccination sera from adults, we analyzed in vitro phagocytic capacities of three different combinations of polymorphonuclear leukocyte FcgammaR allotypes: those homozygous for the H131 and NA1 allotype, those homozygous for the R131 and NA2 allotype, and those heterozygous for both receptors. For pre- and postvaccination sera, mean phagocytosis levels for the homozygous H131/NA1 allotype were 4 -fold higher than for the homozygous R131/NA2 allotype. There was a strong and significant correlation between IgG2 ELISA antibody titers and phagocytosis levels for the homozygous H131/NA1 Fcgamma receptor allotype and the heterozygous allotype but not for the homozygous R131/NA2 allotype. There was no relation between IgG1 ELISA titer and phagocytosis level. Apparently the IgG2 antibodies induced are functionally the most important. This may explain the large effect of Fcgamma receptor polymorphisms on in vitro phagocytosis of pneumococci mediated by conjugate antisera.

Adjuvant Quil A improves protection in mice and enhances opsonic capacity of antisera induced by pneumococcal polysaccharide conjugate vaccines

The adjuvant effect of Quil A on the primary antibody response of mice to pneumococcal capsular polysaccharide conjugates was examined. Quil A increased the anti-capsular polysaccharide antibody titres, the protection against Streptococcus pneumoniae, and the opsonic capacity of the antibodies as measured in a newly developed in vitro phagocytosis assay, using the mouse macrophage cell line J774.

Enhancement of HIV-1 replication in peripheral blood mononuclear cells by Cryptococcus neoformans is monocyte-dependent but tumour necrosis factor-independent.

Objective:To investigate the possible role of Cryptococcus neoformans in HIV-1 pathogenesis. Design:An in vitro system was developed to study HIV-1 replication in freshly HIV-1-infected peripheral blood mononuclear cells (PBMC) incubated with whole azide-killed C. neoformans. Methods:Human PBMC or peripheral blood lymphocytes were infected with lymphocytotropic HIV-1 and incubated with azide-killed encapsulated or non-encapsulated C. neoformans for 10 days. Viral replication was followed by HIV-1 p24 enzyme-linked immunosorbent assay and median tissue culture infective dose determination. Tumour necrosis factor (TNF) release by PBMC, induced by C. neoformans, was measured. Anti-TNF monoclonal antibodies or pentoxifylline were used to inhibit TNF bioactivity. Results:Both encapsulated and non-encapsulated C. neoformans enhanced HIV-1 replication in PBMC but not in peripheral blood lymphocytes. C. neoformans induced TNF release by PBMC. Inhibition of TNF bioactivity did not block C. neoformans-enhanced HIV-1 replication in PBMC. Conclusions:C. neoformans can enhance HIV-1 replication in T cells only in the presence of monocytic cells. This enhancement is not dependent on encapsulation nor can it be attributed to TNF release.

Comparison of a classical phagocytosis assay and a flow cytometry assay for assessment of the phagocytic capacity of sera from adults vaccinated with a pneumococcal conjugate vaccine.

ABSTRACT Antibody- and complement-mediated phagocytosis is the main defense mechanism against Streptococcus pneumoniae. A standardized, easy to perform phagocytosis assay for pneumococci would be a great asset for the evaluation of the potential efficacy of (experimental) pneumococcal vaccines. Such an assay could replace the laborious phagocytosis assay of viable pneumococci (classical killing assay). Therefore, a newly developed phagocytosis assay based on flow cytometry (flow assay) was compared with the conventional killing assay and enzyme-linked immunosorbent assay (ELISA), using sera obtained from adults pre- and postvaccination with either a bivalent conjugate, a tetravalent conjugate, or the 23-valent polysaccharide vaccine. Highly significant correlations were observed between flow assay phagocytosis titers, killing assay phagocytosis titers, and ELISA antibody titers for serotype 6B and 23F as well. For serotype 19F, strong correlations were only observed between killing assay and ELISA titers. A potential drawback of the flow assay might be the low sensitivity compared with that of the killing assay. The choice of what assay to use, however, will depend on the objectives of the assay. When speed, easy performance, sample throughput, improved worker safety, absence of influence of antibiotics, and absence of false positives are the major criteria, the flow assay is the method of choice. When higher sensitivity is the major requirement, the classical killing assay should be used.

Induction of antibody and T-cell responses by immunization with ISCOMS containing the 38-kilodalton protein of Mycobacterium tuberculosis

In this study, we investigated the influence of different amounts of N-(palmitoyloxy) succinimide (PA-NHS): attachment of lipid tails to the protein and Quil A on the immunogenicity of the 38-kDa mycobacterial protein incorporated into immunostimulating complexes (ISCOMS; 38-kDa ISCOMS). The addition of higher amounts of Quil A during the ISCOMS preparation increased the amount of protein incorporated into ISCOMS, whereas the use of higher amounts of PA did not influence this parameter. Low antibody responses were observed after primary immunization with all 38-kDa ISCOMS preparations which, however, strongly increased after booster injections. IgG2a is the major subclass IgG induced by these ISCOMS preparations. There were only slight differences between the various ISCOMS formulations in their capacity to induce cytotoxic T-lymphocytes (CTLs). Spleen cells primed with ISCOMS prepared with the highest amount of Quil A produced high levels of IFN-gamma after stimulation with T helper cell type one (Th1) peptide of the 38-kDa protein (aa 70-84), 38-kDa protein or purified protein derivate (PPD). Spleen cells primed with ISCOMS prepared with the lowest amount of Quil A only substantial IFN-gamma levels were detected after stimulation with 38-kDa protein. IL-4 secretion was very low or not detectable with all ISCOM preparations. These results therefore demonstrated that all 38 kDa-ISCOMS preparations were: (1) immunogenic by inducing antibodies, Th1 and CTL responses; (2) that the way in which the ISCOMS were prepared, e.g. the amount of Quil A used, modulates the epitope specificity of the Th1 response.

Evaluation of T-cell responses to peptides and lipopeptides with MHC class I binding motifs derived from the amino acid sequence of the 19-kDa lipoprotein of Mycobacterium tuberculosis.

Cytotoxic T-lymphocyte (CTL) epitopes on the 19-kDa lipoprotein from Mycobacterium tuberculosis were identified by the use of lipopeptides and their cytokine profile studied. Selection of candidate CTL epitopes was based on synthetic peptides derived from the amino acid sequence of the 19-kDa lipoprotein showing major histocompatibility complex class I (MHC-I) binding motifs (H-2D(b) and H-2L(d)). Their ability to up-regulate and stabilize MHC-I molecules on the mouse lymphoma cell line RMA-S was studied. Similar studies were performed with peptides, in which the anchor amino acid of the H-2D(b) MHC-I motif was replaced by alanine. Three out of five peptides with H-2D(b) or H-2L(d) binding motifs and their corresponding lipopeptides as well, up-regulated and stabilized the H-2D(b) molecules on RMA-S cells. Replacement of the anchor amino acid residues of the H-2D(b) MHC-I motif by alanine revealed that the anchor amino acid asparagine at position 5, contributed more to binding of peptide to H-2D(b) molecules than leucine at position 11. The closely related lipopeptides LP19c and LP19d, in combination with incomplete Freunds adjuvant (IFA), induced CTL responses in C57BL/6 (H-2(b)) mice. These CTLs could recognize the naturally processed antigen, i.e. the 19-kDa antigen protein produced and processed by the EX-19 cell line. The capacity of the various lipopeptides to induce CTL correlated well with the ability of the (lipo)peptide to up-regulate and to stabilize H-2D(b) molecules. Lipopeptide LP19c primed spleen cells showed a T helper type one profile after in vitro stimulation with P19c and P19d 19 kDa peptides. The approach to characterize presumptive 19-kDa CTL epitopes might lead to selection of promising CTL epitopes, which can be applied in the development of subunit tuberculosis vaccines.