Raymond Hamers

Selection and identification of single domain antibody fragments from camel heavy‐chain antibodies

Functional heavy‐chain γ‐immunoglobulins lacking light chains occur naturally in Camelidae. We now show the feasibility of immunising a dromedary, cloning the repertoire of the variable domains of its heavy‐chain antibodies and panning, leading to the successful identification of minimum sized antigen binders. The recombinant binders are expressed well in E. coli, extremely stable, highly soluble, and react specifically and with high affinity to the antigens. This approach can be viewed as a general route to obtain small binders with favourable characteristics and valuable perspectives as modular building blocks to manufacture multispecific or multifunctional chimaeric proteins.

A VSG Expression Site–Associated Gene Confers Resistance to Human Serum in Trypanosoma rhodesiense

Infectivity of Trypanosoma brucei rhodesiense to humans is due to its resistance to a lytic factor present in human serum. In the ETat 1 strain this character was associated with antigenic variation, since expression of the ETat 1.10 variant surface glycoprotein was required to generate resistant (R) clones. In addition, in this strain transcription of a gene termed SRA was detected in R clones only. We show that the ETat 1.10 expression site is the one selectively transcribed in R variants. This expression site contains SRA as an expression site-associated gene (ESAG) and is characterized by the deletion of several ESAGs. Transfection of SRA into T.b. brucei was sufficient to confer resistance to human serum, identifying this gene as one of those responsible for T.b. rhodesiense adaptation to humans.

Camel heavy-chain antibodies: diverse germline VHH and specific mechanisms enlarge the antigen-binding repertoire

The antigen‐binding site of the camel heavy‐chain antibodies devoid of light chain consists of a single variable domain (VHH) that obviously lacks the VH–VL combinatorial diversity. To evaluate the extent of the VHH antigen‐binding repertoire, a germline database was constructed from PCR‐amplified VHH/VH segments of a single specimen of Camelus dromedarius. A total of 33 VHH and 39 VH unique sequences were identified, encoded by 42 and 50 different genes, respectively. Sequence comparison indicates that the VHHs evolved within the VH subgroup III. Nevertheless, the VHH germline segments are highly diverse, leading to a broad structural repertoire of the antigen‐binding loops. Seven VHH subfamilies were recognized, of which five were confirmed to be expressed in vivo. Comparison of germline and cDNA sequences demonstrates that the rearranged VHHs are extensively diversified by somatic mutation processes, leading to an additional hypervariable region and a high incidence of nucleotide insertions or deletions. These diversification processes are driven by hypermutation and recombination hotspots embedded in the VHH germline genes at the regions affecting the structure of the antigen‐binding loops.

THE SERUM RESISTANCE-ASSOCIATED (SRA) GENE OF TRYPANOSOMA BRUCEI RHODESIENSE ENCODES A VARIANT SURFACE GLYCOPROTEIN-LIKE PROTEIN

In the Trypanosoma brucei species, T. b. rhodesiense and T. b. gambiense represent the human infective host range variants, while T. b. brucei is lysed upon exposure to a cytotoxic factor in normal human serum. T. b. rhodesiense can occur in a serum-resistant and a serum-sensitive form. The resistance towards normal human serum was shown to be a labile character and to be determined by the environment in which the parasites live. We have clearly demonstrated the presence of RNA transcripts unique to the resistant forms of T. b. rhodesiense. These transcripts encode a protein with VSG characteristics. The DNA fragment isolated previously, which hybridises with the resistance-specific mRNA sequence, appears to be a pseudogene belonging to the same gene family.

Loss of splice consensus signal is responsible for the removal of the entire CH1 domain of the functional camel IGG2A heavy-chain antibodies

The molecular basis for the absence of the C(H)1 domain in naturally occurring heavy-chain antibodies of the camelids was assessed by determining the entire Camelus dromedarius gamma2a heavy-chain constant gene. The organization of the camel gamma2a constant heavy-chain gene obtained from a liver genomic library appears to be typical of all other mammalian gamma genes sequenced to date. It contains the switch, CH1, hinge, CH2, CH3, M1 and M2 exons. In contrast to the case in mouse and human heavy chain diseases, the camel gamma2a gene shows no major structural defect, and its equivalent CHI exon is intact. However, sequence analysis has revealed that the splicing site, immediately after the CH1 exon, is defective due to point mutations, especially the G(+1) to A(+1) transversion seems to be detrimental. It is concluded that the loss of the splice consensus signal is responsible for the removal of the entire CH1 domain in camel gamma2a heavy-chain immunoglobulins. Additionally, a closer analysis of the hinge exon suggests the possible involvement of transposons in the genetic variation of mammalian Cgamma hinges.

A gene expressed only in serum-resistant variants of Trypanosoma brucei Rhodesiense

The human infective African trypanosomes are host range variants of Trypanosoma brucei which are resistant to a lytic component in primate serum. T. b. rhodesiense occurs both as a form sensitive to lysis by normal human serum and as a form resistant to this lysis. Switching from one phenotype to the other has been observed in both directions. In the cloned T. b. rhodesiense ETAR1-repertoire we have detected 1.5-kb mRNAs only present in the resistant forms. In T. b. gambiense, which always occurs as a normal human serum-resistant form, no such transcript could be detected, indicating that another mechanism of resistance is involved here. Starting from an independent non-cloned T. b. rhodesiense population isolated from an infected patient, both resistant and sensitive trypanosomes have been prepared. Northern blot analysis of the total RNA prepared from these populations has revealed again the differential occurrence of the resistance-specific transcript, indicating that we are dealing with a general phenomenon associated with serum resistance in T. b. rhodesiense. As expected, Southern blot analyses have demonstrated that both serum-resistant and serum-sensitive forms of T. b. rhodesiense contain the gene coding for this transcript.

Trypanosoma (Nannomonas) congolense: identification of two karyotypic groups.

Orthogonal‐field‐alternation gel electrophoresis and DNA blot hybridizations have been used to investigate the genomic relationships among trypanosome clones of subgenus Nannomonas. The results indicate that Trypanosoma (Nannomonas) congolense comprises at least two distinct groups of parasites that differ in both molecular karyotype and repetitive DNA sequences. A description of these two groups and their distinction from Trypanosoma (Nannomonas) simiae is presented.

Murine tumour necrosis factor plays a protective role during the initial phase of the experimental infection with Trypanosoma brucei brucei

Soluble extracts from salivarian trypanosomes (Trypanosoma brucei brucei, T. evansi and T. congolense) were shown to be capable of inducing murine tumour necrosis factor (mTNF) secretion, both in vivo and in vitro, whereas the soluble extract of an intracellular trypanosome (T. cruzi) failed to do so. Furthermore, the role of mTNF during the initial phase of experimental infections with T. brucei was studied by treating infected mice with mTNF‐inducing trypanosoma soluble extract and with neutralizing monoclonal anti‐mTNF antibodies. Treatment of the infected animals with different doses of T. brucei soluble extract resulted in a lower first parasitaemia peak (low lysate dose) and in a longer survival time or in a nearly total inhibition of parasite development (high lysate dose). Cotreatment of the infected mice with both anti‐mTNF antibodies and a high dose of soluble extract completely restored the parasite development in both trypanosusceptible C3H/He mice and trypanosubtolerant CBA/Ca mice, indicating a protective role of mTNF during the parasitaemia. Collectively these results suggest a negative influence of mTNF on T. brucei development in vivo.

Plasticity of gp63 gene organization in Leishmania (Viannia) braziliensis and Leishmania (Viannia) peruviana.

The genomic organization of gp63 genes in 4 and 7 isolates of Leishmania braziliensis and L. peruviana, respectively was studied by RFLP analysis with 3 restriction enzymes (Bgl I, Sal I and Apa I). Our results showed a marked polymorphism among isolates. Some characters were specific to L. braziliensis or to L. peruviana, and others specific to the respective biogeographical populations of L. peruviana. The average minimum copy number of gp63 genes was found to be higher in L. braziliensis (71) than in L. peruviana (46), suggesting that deletion of gp63 genes might be partially involved in the size decrease of the chromosome bearing gp63 genes, observed between those 2 species (from 700 to 610 kb). Our results may suggest the existence of at least 2 arrays of heterologous gp63 repeats, varying in relative copy number between L. braziliensis and L. peruviana, and among isolates of the latter species. Rearrangement of the gp63 genes was observed during long-term in vitro maintenance of a reference strain of L. braziliensis. These observations document the existence of a dynamic gp63 gene organization in Leishmania of the braziliensis complex.

Molecular cloning and sequence analysis of the gene encoding the major merozoite surface antigen of Plasmodium chabaudi chabaudi IP-PC1.

The complete nucleotide sequence of the gene encoding the precursor to the major merozoite surface antigens of Plasmodium chabaudi chabaudi strain IP-PC1 has been determined. A single open reading frame was detected, that coded for a protein of 199 kDa. The encoded protein (p199) contains putative signal and membrane anchor sequences and shows a clustering of Cys residues in the last 120 amino acids. Incompletely conserved tandem repeat oligopeptides are present at different positions in the molecule. P199 shows 69% overall homology to the analogous antigen in Plasmodium yoelii yoelii strain YM. The divergence between these antigens is largely confined to 4 areas where a number of insertions and/or deletions have occurred. All repeats occur in these divergent regions. The overall homology with both alleles of Plasmodium falciparum PMMSA is 33%.