Characterization of human CD4(+) T-cell clones recognizing conserved and variable epitopes of the Lassa virus nucleoprotein

Abstract

T cells must play the major role in controlling acute human Lassa virus infection, because patients recover from acute Lassa fever in the absence of a measurable neutralizing antibody response. T cells alone seem to protect animals from a lethal Lassa virus challenge, because after experimental vaccination no neutralizing antibodies are detectable. In order to study human T-cell reactivity to single Lassa virus proteins, the nucleoprotein (NP) of Lassa virus, strain Josiah, was cloned, expressed in Escherichia coli, and affinity purified. Peripheral blood mononuclear cells (PBMC) obtained from 8 of 13 healthy, Lassa virus antibody-positive individuals living in the Republic of Guinea, western Africa, were found to proliferate in response to the recombinant protein (proliferation index >/=10). PBMC obtained from one individual with a particularly high proliferative response were used to generate 50 NP-specific T-cell clones (TCC). For six of these the epitopes were mapped with overlapping synthetic peptides derived from the sequence of the NP. These CD4(+) TCC displayed high specific proliferation and produced mainly gamma interferon upon stimulation with NP. Because variation of up to 15% in the amino acid sequences of the structural proteins of naturally occurring Lassa virus variants has been observed, the reactivity of the TCC with peptides derived from the homologous epitopes of the Nigeria strain of Lassa virus and of the eastern Africa arenavirus Mopeia was tested. With the Nigeria strain of Lassa virus the levels of homology were 100% for two of these epitopes and 85% for three of them, whereas homology with the respective Mopeia epitopes ranged from 92 to 69%. Reactivity of the TCC with peptides derived from the variable epitopes of the Nigeria strain and of Mopeia was reduced or completely abolished. This report shows for the first time that seropositive individuals from areas of endemicity have very strong memory CD4(+) T-cell responses against the NP of Lassa virus, which are partly strain specific and partly cross-reactive with other Lassa virus strains. Our findings may have important implications for the strategy of designing recombinant vaccines against this mainly T-cell-controlled human arenavirus infection.

FIG. 1

Proliferative response of PBMC to pooled stimulatory peptides. Assays were run in duplicate (two wells). Each peptide pool (PP) contains six overlapping peptides consisting of 20 aa. PP1 to PP10 span the whole recNP of Lassa virus JOS. Specific proliferation was assessed by calculating [³H]thymidine uptake after a 3-day culture and is expressed as PI (see Materials and Methods). Only statistically significant proliferations are shown (see Materials and Methods). Epitopes of TCC of the TCC donor are located in the overlap of the respective adjacent peptides (Fig. 3), which are contained in the respective peptide pools. ■, donor; ▨, subject 10.2.22; ▥, subject 8.1.10.

FIG. 2

Proliferation assays of TCC 148 with different stimuli and measurement of cytokines. Assays were run in duplicate (two wells each); the results of one of two consistent experiments are shown. (A) Proliferation ([³H]thymidine counts per minute) in response to PHA, recNP, peptide pools (PP; containing six stimulatory, overlapping peptides each), and individual peptides. Blocking of proliferation was with anti-DR but not anti-DQ MAbs. (B and C) Production of IFN-γ and IL4 in response to specific and nonspecific stimuli. Error bars, two standard deviations.

FIG. 3

Alignment of JOS NP CD4 T-cell epitopes with heterologous arenaviruses. Boxed regions indicate overlap of stimulatory peptides; the numbering of peptides is according to Table 2. Amino acids in boldface differ from those in the JOS NP sequence. Numbers below boxes indicate the positions of the first and last amino acids, as in Lassa virus, strain JOS (EMBL accession no., JO 4324). Sequences of NIG and MOP have EMBL accession no. X52400 and M33879, respectively.

FIG. 4

Reactivity of TCC 141 and 148 to stimulatory peptides from JOS and homologous peptides derived from the sequences of NIG and MOP. Assays were run in triplicate; the representative results of one of three independent experiments are shown. Error bars, two standard deviations. Reactivity of TCC 135 to stimulatory peptides from JOS and homologous peptides derived from the sequences of NIG and MOP. Assays were run in duplicate; the representative results of one of two independent experiments are shown. Error bars, two standard deviations. Amino acids in bold type differ form the Josiah sequence.