Immune response to native NadA from Neisseria meningitidis and its expression in clinical isolates in Brazil

A mAb against the NadA protein from Neisseria meningitidis strain 3006 (serosubtype B : 2b : P1.2 : P5.2,8) demonstrated strong bactericidal activity against Brazilian epidemic serogroup B strain N44/89 (B : 4,7 : P1.19,15 : P5.5,7) and a serogroup C strain, IMC 2135 (C : 2a : P1.5,2), but not against another serogroup C strain, N1002/90 (C : 2b : P1.3 : P5.8). The immunogenicity of native NadA in an outer-membrane vesicle (OMV) preparation was also tested. Serum from mice immunized with OMV from serogroup B strain N44/89, which contains the NadA protein, showed bactericidal activity against serogroup B and C strains possessing NadA. In dot-blot analysis of 100 serogroup B and 100 serogroup C isolates from Brazilian patients, the mAb to NadA recognized about 60% of the samples from both serogroups. The molecular mass of the NadA protein from strain N44/89 determined by mass spectrometry was 37 971 Da and the peptide sequences were identical to those of NadA from N. meningitidis strain MC58.

PsaA (pneumococcal surface adhesin A) and PspA (pneumococcal surface protein A) DNA vaccines induce humoral and cellular immune responses against Streptococcus pneumoniae.

Streptococcus pneumoniae is one of the most important human pathogens and improvement of the currently used polysaccharide vaccines is being pursued. We constructed DNA vaccine vectors containing either the full-length psaA (pneumococcal surface adhesin A) or a truncated pspA (pneumococcal surface protein A--pspA') gene. Both constructs showed transient expression of the antigens in vertebrate cells and induced significant antibody response to the pneumococcal antigens in BALB/c mice injected intramuscularly (i.m.). Fusion with an N-terminal cytoplasmatic SV40 T-antigen (CT-Ag), which was previously shown to stabilize poorly expressed antigens through association with Hsp73, also induced anti-PspA antibody response. The induction of antibodies with a low IgG1:IgG2a ratio and elevated gamma interferon (IFN-gamma) production by spleen cells elicited by DNA vaccination indicate preferential priming of Th1 immunity. Since induction of antibodies against both PsaA and PspA was previously shown to correlate with protection against fatal infection with S. pneumoniae and cell-mediated immune responses could contribute to protection, further evaluation of PsaA and PspA as antigens for a DNA vaccine against S. pneumoniae could be promising.

Neisseria meningitidis serogroup C polysaccharide and serogroup B outer membrane vesicle conjugate as a bivalent meningococcus vaccine candidate.

Neisseria meningitidis serogroup C polysaccharide (PS C) was conjugated to serogroup B outer membrane vesicles (OMV) in order to test the possibility of obtaining a bivalent group B and C meningococcus vaccine. The conjugate and controls were injected intraperitoneally into groups of ten mice with boosters on days 14 and 28 after the primary immunization. The following groups were used as control: (i) PS C; (ii) PS C plus OMV; (iii) OMV; and (iv) saline. The serum collected on days 0, 14, 28 and 42 were tested by enzyme-linked immunosorbent assay (ELISA) for PS C and OMV, and by complement mediated bactericidal assay against serogroups B and C. ELISA for PS C as well as bactericidal titres against serogroup C meningococci of the conjugated vaccine increased eight-fold (ELISA) and 32 fold (bactericidal) after 42 days in comparison with the PS C control group. ELISA for OMV and bactericidal titre against serogroup B meningococci of the conjugate showed no significant difference in comparison with the OMV containing controls. Furthermore, Western Blot assay of the conjugate immune serum did not bind OMV class four protein which is related to the complement dependent antibody suppressor. The results indicate that the PS C-OMV conjugate could be a candidate for a bivalent vaccine toward serogroups B and C meningococci.

Organization of trans-sialidase genes in Trypanosoma cruzi.

Trypanosoma cruzi trans-sialidase is encoded by a family of genes containing a conserved region, which corresponds to the catalytic and amino-terminal domain of the enzyme. Most, but not all genes, also encode a variable region formed by 12 amino acid repeats at the carboxy-terminus of the protein that are not required for enzymatic activity. To design gene knock-out strategies and understand how trans-sialidase expression is regulated, we have studied the genome organization of trans-sialidase genes. We show here that the different types of trans-sialidase genes are distributed in more than one chromosomal band with sizes ranging from 0.8 to 1.5 Mb pairs in several T. cruzi strains. In the Y-strain, all repeat-containing genes are localized in one chromosomal band of 1.1 Mb, while the repeat-minus genes are in two chromosomes of 0.82 and 0.79 Mb. The repeat-containing genes have similar catalytic and intergenic regions, but variable lengths of the repeated region. The trans-sialidase genes with the repeats are in tandem of up to 12 genes in at least four different clusters. Each cluster contains genes with different numbers of repeats, according to the physical maps of eight independent cosmids, and in the same cluster there are genes that code for active and inactive trans-sialidases. There are 80 +/- 30 copies of the repeat-containing genes grouped in two NotI fragments of 120 and 180 Kb. Therefore, in the Y-strain, the trans-sialidase genes containing repeats might be arranged in three to four clusters in two homologous chromosomes, each cluster having up to 12 genes with different repeat numbers.

Sialic acid acceptors of different stages of Trypanosoma cruzi are mucin-like glycoproteins linked to the parasite membrane by GPI anchors.

Trans-sialidase catalyzes the transference of sialic acid from host to the Trypanosoma cruzi surface. Here, we characterize the sialic acid acceptors of this protozoan parasite as mucin-like molecules, which are anchored to the membrane by glycosylphosphatidylinositol. The mucins isolated from the insect stages differ from the mucins isolated from the mammalian stages in size and reactivity to monoclonal antibodies, suggesting that they are formed by variable polypeptide chains and/or O-linked carbohydrate structures.

Mammalian cell sialic acid enhances invasion by Trypanosoma cruzi.

We have used a Chinese hamster ovary cell mutant (Lec2) that express much less sialic acid on the surface than the parental cell line (Pro5) to investigate whether sialic acid plays a role during cell invasion by Trypanosoma cruzi. Trypomastigotes derived from a tissue culture (corresponding to bloodstream trypomastigotes) and metacyclic trypomastigotes (corresponding to infective stages of the insect vector) invaded the Lec2 mutant less efficiently than the parental cell line. Invasion of the Lec2 mutant cells could be restored to the Pro5 level by resialylation of the mutant cells with T. cruzi trans-sialidase and sialyllactose. Conversely, pretreatment of the Pro5 parental cells with bacterial neuraminidase decreased invasion. These results indicate that sialic acid associated with the host cell contributes to invasion by T. cruzi.