T-cell-stimulating protein A elicits immune responses during meningococcal carriage and human disease

Abstract

In recognition of the need for immunological memory-inducing components for future Neisseria meningitidis group B vaccines, we previously searched the proteome of N. meningitidis and identified T-cell-stimulating protein A (TspA). This study was designed to confirm the immunogencity of TspA and to examine the subset of T-helper cell responses to the protein in patients and nasopharyngeal carriers. The tspA gene was reconstructed, cloned, and expressed in Escherichia coli, and the recombinant TspA (rTspA) protein was affinity purified. T-cell proliferative responses to rTspA were detected in the peripheral blood mononuclear cells (PBMCs) of convalescent patients and carriers, confirming that TspA-specific T-cell responses were stimulated by invasive disease and nasopharyngeal colonization. Following stimulation of PBMCs with meningococcal lysate, increased frequencies of both Th1 and Th2 cells were observed, indicating that, as during carriage, invasive meningococcal disease induced an unbiased T-helper subset response. A similar unbiased T-helper response was also detected against rTspA in the PBMCs of convalescent patients. The response of PBMCs from the carriers to TspA stimulation, however, was very weak, and the frequencies of cytokine-positive CD4 cells were not significantly greater than the frequencies in unstimulated control cultures. All of the patients and carriers responded with serum antimeningococcal immunoglobulin G (IgG) antibodies, while four of six samples from patients and 5 of 14 samples from carriers contained detectable anti-rTspA IgG antibodies. Taken together, the results of this study confirmed the immunogenicity of TspA in humans during natural meningococcal infection, and therefore, TspA is worthy of further investigation as a possible T-cell stimulating component of future vaccines.

FIG. 1.

Gel electrophoresis protein profiles following expression of recombinant TspA: SDS-PAGE gel showing protein profiles of the control E. coli expression strain lysate (lane 1) and E. coli containing the TspA expression vector following induction to express TspA (lane 2). The positions of TspA and molecular weight markers (in kDa) are indicated on the right and left, respectively.

FIG. 2.

Proliferative responses of PBMCs to optimum stimulatory concentrations of a heat-killed whole-cell lysate of the H44/76 meningococcal strain, recombinant TspA, a negative control TspA preparation, or tetanus toxoid. Replicate wells of PBMCs were incubated with 1 to 10 μg/ml of the H44/76 strain lysate, 0.5 to 5 LfU/ml of tetanus toxoid (TT), 0.1 to 5 μg/ml of TspA, and equivalent volumes of a negative control preparation from the E. coli host strain used for TspA expression. PHA (10 μg/ml) was added as a positive control, while other wells received no antigen or mitogen. Cells were cultured for 7 days, and [³H]thymidine was added for the final 18 h. SIs were calculated by determining the ratio of mean counts per minute from antigen-stimulated cells to mean counts per minute from unstimulated cells. The data show the SIs obtained from the PBMCs of individual convalescent patients (a) and confirmed carriers (b). The bars indicate the geometric mean SIs for the groups, while the error bars indicate the standard deviations. The stimulation indices obtained from induction with PMA were greater than 139.2.

FIG. 3.

Frequencies of activated CD4 cells staining positive for IFN-γ or IL-5 following incubation of PBMCs from patients (a) and carriers (b) for 10 h with a lysate of the H44/76 meningococcal strain or recombinant TspA. The data points indicate the individual frequencies of cytokine-positive CD69⁺ CD4⁺ events. The lines indicate the median frequencies for groups. (c) Proportion of IFN-γ- or IL-5-producing cells which expressed CD4, CD8, or CD56 for PBMCs from patients. The solid bars indicate the geometric mean response to the meningococcal lysate, and the open bars indicate the response to rTspA; the error bars indicate the standard deviations.

FIG. 4.

Levels of antimeningococcal (a) and anti-TspA (b) IgG in serum samples from 14 carriers and six patients recovering from meningococcal disease (Conv. Patient). The bars indicate the geometric mean concentrations; the error bars indicate the standard deviations.

FIG. 5.

Detection of TspA-specific antibody in serum from convalescent patients (P1 to P6) and confirmed meningococcal carriers (C1 to C8). Recombinant TspA was loaded onto a 10% SDS-PAGE gel before electrophoretic transfer to nitrocellulose. The membrane was cut into strips and probed in immunoblotting experiments with human sera, followed by anti-human IgG-alkaline phosphatase conjugate. Positive controls (+) were probed with rabbit anti-TspA serum and an anti-rabbit IgG conjugate. Negative controls (−) were incubated only with the anti-human IgG conjugate antibody.