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Display-induced antigenic variation in recombinant peptides.

Peptide display on solvent-exposed surfaces of carrier proteins is a promising approach pursuing the identification and improvement of reactive amino acid sequences. However, the contribution of the molecular environment where the peptide is inserted on its interactive properties remains essentially unexplored. By an exhaustive antigenic analysis of the same peptide displayed on 20 structurally distinct frameworks, we show that peptide accommodation into the acceptor site has dramatic effects on its immunoreactivity. Conformational constraints can modulate the molecular recognition properties of the insert within a surprisingly wide range, probably by affecting the positioning of critical contact residues. The observed display-induced antigenic variation prompts a careful consideration of the molecular context when evaluating output amino acid sequences from screening of peptide libraries or application of directed molecular evolution technologies.

Amino Acid Sequence↗

Genetic and antigenic variations among geographical isolates of Sindbis virus.

The genetic and antigenic variation in 12 Sindbis (SIN) virus isolates from four zoogeographic regions (Paleoarctic, Ethiopian, Oriental and Australian) has been examined at a molecular level. RNase T1 oligonucleotide fingerprinting of genomic RNA from SIN isolates revealed that the primary structure of the RNA from viruses from each zoogeographic region was unique. The E1 and E2 glycoproteins and the capsid protein of two isolates from each zoogeographic region were compared by tryptic peptide mapping with the Egyptian prototype strain AR-339. Tryptic peptide maps of viruses from Sicily and the Ethiopian region were similar to those of the prototype; maps of isolates from the Oriental and Australia regions were different from each other and from those of the prototype strain. Viruses from each of the four zoogeographic regions were analysed antigenically by neutralization with polyclonal serum to AR-339 and by enzyme-linked immunosorbent assay with an anti-E2 monoclonal AR-339 antibody. Clear antigenic divergence of SIN isolates into two groups, representing the Paleoartic-Ethiopian and Oriental-Australian regions were demonstrated. These results support a hypothesis which proposes that ancestral SIN virus diverged into two distinct groups. The genetic changes have resulted in further phenotypic divergence within the geographic varieties.

Amino Acid Sequence↗

Monoclonal antibodies define antigenic variation in the ID variety of Venezuelan equine encephalitis virus.

Three monoclonal antibodies were generated that are specific for the E2 glycoprotein of Venezuelan equine encephalitis (VEE) virus and have useful reactivities in an enzyme-linked immunosorbent assay (ELISA). Antibody 1A1B-9 distinguished between the IC (epizootic) and ID (enzootic) varieties of VEE virus by ELISA. Clone 7A1A-1 antibody distinguished the Panamanian prototype virus (3880) from Colombian ID isolates by a 500-fold difference in titer by endpoint ELISA, and it detected antigenic variation in ID isolates from southern Colombia and Ecuador. Antibody 7A3A-4 defined a cryptic antigenic site on the latter two isolates. These monoclonal antibodies complement others in identifying VEE isolates by a simple ELISA.

Animals↗

Intragenic recombination leads to pilus antigenic variation in Neisseria gonorrhoeae.

The pilus of the bacterium Neisseria gonorrhoeae is a fimbriate surface structure which promotes attachment of the bacterium to host epithelial cells. Gonococcal pilus phase variation is characterized by a rapid on/off switch in which piliated (P+) cells throw off non-piliated (P-) variants and vice versa. Two regions of the gonococcal chromosome (pilE1 and pilE2) act as pilin expression loci, reminiscent of the MAT locus in the yeast Saccharomyces cerevisiae, while several other chromosomal regions contain silent (non-expressing) pilin sequences. Biochemical and antigenic diversity is seen in pili from a wide variety of clinical isolates. Pilins (pilus subunits) are composed of conserved N-terminal and variable C-terminal regions; the conserved region of gonococcal pilin is also found in pilins produced by widely disparate bacteria. We show here that the gonococcal pilin undergoes antigenic variation in vitro and in vivo. The protein consists of constant, semi-variable and hypervariable regions. This antigenic variation probably involves gene conversion of mini-cassettes of pilin information.

Amino Acid Sequence↗

In vivo antigenic variation of Campylobacter flagellin.

Campylobacter coli VC167 cells producing either antigenic phase 1 (P1) or phase 2 (P2) flagellins (as determined by characteristic protein and DNA patterns) were used to infect rabbits by the removable intestinal tie-adult rabbit diarrhea (RITARD) procedure. Rabbits infected with P2 cells shed predominantly P2 cells throughout the infection; in rabbits infected with P1 cells, a transition of fecal isolates from P1 to P2 was observed.

Animals↗

Antigenic variation during persistent infection by equine infectious anemia virus, a retrovirus.

The recurrent nature of equine infectious anemia has been attributed to relatively rapid antigenic variations in equine infectious anemia virus (EIAV) during persistent infection under selective immune pressures. This model was tested by serological and biochemical analysis of virus isolates recovered from separate febrile episodes in two experimentally infected ponies. Neutralization assays employing immune sera from the experimentally infected ponies demonstrated that distinct antigenic strains of virus predominate during sequential febrile episodes in a single pony. Analysis of the test strains of EIAV by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed varying electrophoretic mobilities for the respective virion glycoproteins gp90 and gp45. Furthermore, peptide mapping comparisons demonstrated structural variations between the gp90 components of the various strains. In contrast, the respective internal proteins of the virus strains displayed identical electrophoretic mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and, in general, produced identical tryptic peptide maps. The observed differences in glycoprotein electrophoretic mobility and peptide maps were highly reproducible and did not vary with repeated passage of the virus strains in cell culture. Thus, these results demonstrate the occurrence of glycoprotein-specific structural variations during persistent infection by EIAV and support the concept of antigenic variation in this retrovirus. This capacity to alter envelope glycoprotein structure, previously reported for visna virus, may represent an important mechanism of retrovirus persistence in the presence of immune responses by the animal host.

Animals↗

Transcriptome analysis of antigenic variation in Plasmodium falciparum--var silencing is not dependent on antisense RNA.

BACKGROUND: Plasmodium falciparum, the causative agent of the most severe form of malaria, undergoes antigenic variation through successive presentation of a family of antigens on the surface of parasitized erythrocytes. These antigens, known as Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) proteins, are subject to a mutually exclusive expression system, and are encoded by the multigene var family. The mechanism whereby inactive var genes are silenced is poorly understood. To investigate transcriptional features of this mechanism, we conducted a microarray analysis of parasites that were selected to express different var genes by adhesion to chondroitin sulfate A (CSA) or CD36. RESULTS: In addition to oligonucleotides for all predicted protein-coding genes, oligonucleotide probes specific to each known var gene of the FCR3 background were designed and added to the microarray, as well as tiled sense and antisense probes for a subset of var genes. In parasites selected for adhesion to CSA, one full-length var gene (var2csa) was strongly upregulated, as were sense RNA molecules emanating from the 3' end of a limited subset of other var genes. No global relationship between sense and antisense production of var genes was observed, but notably, some var genes had coincident high levels of both antisense and sense transcript. CONCLUSION: Mutually exclusive expression of PfEMP1 proteins results from transcriptional silencing of non-expressed var genes. The distribution of steady-state sense and antisense RNA at var loci are not consistent with a silencing mechanism based on antisense silencing of inactive var genes. Silencing of var loci is also associated with altered regulation of genes distal to var loci.

Animals↗

Antigenic variation and cytoadhesion in Babesia bovis and Plasmodium falciparum: different logics achieve the same goal.

Babesia bovis is a protozoal hemoparasite of cattle which behaves in certain crucial respects like Plasmodium falciparum, despite being phylogenetically distant and having many differences in its life cycle. The shared behavioral attributes of rapid antigenic variation and cytoadhesion/sequestration are thought to contribute significantly to immune evasion, establishment of persistent infections, and disease pathology. Although differing in their genetic and biochemical strategies for achieving these behaviors, information from studies of each parasite may further our understanding of the overall host-parasite interaction. In this review we contrast the molecular basis and 'genetic logic' for these critical behaviors in the two parasites, with emphasis on the biology of B. bovis.

Animals↗

Antigenic variation in the persistence and transmission of the ehrlichia Anaplasma marginale.

Tick-borne transmission of ehrlichial pathogens requires rickettsemic reservoir hosts to maintain a population of infected vectors. Persistence in their respective mammalian hosts appears to be a common feature of the tick-transmitted ehrlichiae. How infection persists in immunocompetent hosts is unknown. In this review, we describe studies on Anaplasma marginale, an ehrlichial pathogen of cattle, that support antigenic variation as a primary mechanism of persistence.

Amino Acid Sequence↗

Unstable antigenic variation of leptospiras.

The antigenic variants which were found in leptospiras, Leptospira interrogans serovar copenhageni Shibaura grown in a liquid medium containing a homologous antiserum were found to be unstable. The unstable variants showed decreased agglutinability against the homologous antiserum. The decreased agglutinability of the unstable variants reverted to the parent's level of agglutinability after 1 or 2 passages through the normal serum medium. The unstable variants differed antigenically from the parent in the agglutinin-absorption test; however, the agglutinin-absorption test was found to be inadequate for showing the antigenic difference between the parent and the unstable variants because their difference was diminished when the agglutinin-absorbed antigen was increased. The application of Laurell rocket immunoelectrophoresis (LRI) to the SDS-extracted antigens revealed the distinct difference between the parent and the unstable variants. Other findings presented herein indicated the difference between the antigens of the parent and the unstable variants to be one of quantity. The magnitude of the quantitative difference between the antigens could be modified by the medium containing various concentrations of the homologous antiserum. The clones of the unstable variant showed antigenic characteristics which were similar to the original unstable variant.

Agglutination Tests↗

The ves multigene family of B. bovis encodes components of rapid antigenic variation at the infected erythrocyte surface.

B. bovis, an intraerythrocytic protozoal parasite, establishes chronic infections in cattle in part through rapid variation of the polymorphic, heterodimeric VESA1 protein on the infected erythrocyte surface and sequestration of mature parasites. We describe the characterization of the ves1 alpha gene encoding the VESA1a subunit, thus providing a description of a gene whose product is involved in rapid antigenic variation in a babesial parasite. This three-exon gene, a member of a multigene family (ves), encodes a polypeptide with no cleavable signal sequence, a single predicted transmembrane segment, and a cysteine/lysine-rich domain. Variation appears to involve creation and modification or loss of a novel, transcribed copy of the gene.

Amino Acid Sequence↗

Successive synovial Mycoplasma hominis isolates exhibit apparent antigenic variation.

The expression of surface proteins by 14 successive Mycoplasma hominis isolates obtained from the synovial fluid of a chronically infected septic arthritis patient was examined. Marked differences in the expression of surface proteins, as determined by monoclonal antibodies raised against the first isolate, were observed. However, identical restriction patterns and virtually identical hybridization patterns with probes containing the conserved genes of the Mycoplasma capricolum rRNA operon and the Escherichia coli elongation factor Tu suggest that the protein differences might reflect antigenic variation by M. hominis during infection.

Antibodies, Monoclonal↗

Protein shift and antigenic variation in the S-layer of Campylobacter fetus subsp. venerealis during bovine infection accompanied by genomic rearrangement of sapA homologs.

Campylobacter fetus subsp. venerealis isolated from a case of human vaginosis was inoculated into the uterus of a C. fetus-negative heifer. Isolates obtained weekly from the vaginal mucus exhibited variations in high-molecular-mass-protein profiles from that of the original inoculum, which had a dominant 110-kDa S-layer protein. Immunoblots of the weekly isolates with monoclonal antibody probes against the 110-kDa S-layer protein and other C. fetus S-layer proteins demonstrated antigenic shifts. Genomic digests of the isolates probed with a 75-mer oligonucleotide of the conserved sapA region also indicated that antigenic variation of the S-layer is accompanied by DNA rearrangement.

Animals↗

Plasma TAFI antigen variations in healthy subjects.

The Thrombin Activatable Fibrinolysis Inhibitor (TAFI) is a recently described inhibitor of fibrinolysis. The physiological variations of plasma TAFI antigen are not well known. We studied TAFI antigen values in healthy populations with a commercially available kit from Milan Analytica (Switzerland). Broad range of TAFI antigen values (from 41% to 259%) was found in a population of 249 healthy individuals. Gender as well as pregnancy did not influence mean values of TAFI antigen. There was a positive correlation between TAFI antigen and age in female (r = 0.28; p <0.05) but not in male populations. Mean TAFI antigen value of a black African male group [mean +/- SD (range): 87 +/- 23 (39-144%)] was significantly lower than the one of age matched Caucasian men [114 +/- 34 (52-259%)] (p <0.0001). TAFI antigen values were very stable within individuals, they did not significantly vary on day time or at several months period. Thus, in contrast to large inter-individual variations, TAFI antigen levels are quite stable within individuals.

Adult↗

A real-time semi-quantitative RT-PCR assay demonstrates that the pilE sequence dictates the frequency and characteristics of pilin antigenic variation in Neisseria gonorrhoeae.

A semi-quantitative real-time RT-PCR assay was designed to measure gonococcal pilin antigenic variation (SQ-PCR Av assay). This assay employs 17 hybridization probe sets that quantitate subpopulations of pilin transcripts carrying different silent pilin copy sequences and one set that detects total pilE transcript levels. Mixtures of a DNA standard carrying the silent copy being detected and a clone encoding the starting pilE sequence, which is the majority pilE template, provided amplification curves that closely matched the experimental data and allowed an analysis of the contribution of different silent pilin copies to variation. The SQ-PCR Av assay was verified using DNA sequence analysis to demonstrate that this methodology allowed an accurate analysis of pilin variation. Both assays showed that with a specific starting pilE sequence, only a subset of the silent pilin copies recombine into pilE at a detectable level, and that this limited subset was reproducibly detected in replicate cultures. When an isogenic pilE sequence variant was examined using both assays, a new subset of silent copy sequences were detected recombining into pilE and the overall frequency of variation was increased. Thus, the parental pilE sequence influences the frequency of variation and the repertoire of pilin variants produced.

Antigenic Variation↗

[Mechanisms of antigenic variation in influenza virus].

Human influenza A viruses evolve rapidly by antigenic shift and antigenic drift. Antigenic shift occurs by gentic reassortment between currently circulating human viruses and influenza viruses of other origin and by re-emergence of a previously circulating virus. The segmental structure of the virus genome enables reassortment in multiply infected cells and also promotes multiple infection because it results in yielding noninfectious particles which randomly lack some genome segment and only become infectious by complementation with others. Antigenic drift is due to an accumulation of nonsynonymous substitutions in the genes encoding the HA and NA proteins. The limited nature of infection to the respiratory epithelium which is the border of the immune system capacitates the virus to reinfect and grow under the partial immune pressure which results in selecting and expanding antigenic mutants.

Antigenic Variation↗

The Vlp system of Mycoplasma hyorhinis: combinatorial expression of distinct size variant lipoproteins generating high-frequency surface antigenic variation.

Isogenic populations of Mycoplasma hyorhinis undergo in vitro high-frequency phase variation in the expression of surface lipoproteins; these products also vary markedly in size through changes in periodic protein structure (R. Rosengarten and K.S. Wise, Science 247:315-318, 1990). In this report, we rigorously define three distinct translation products comprising the Vlp (variable lipoprotein) system of M. hyorhinis SK76 and establish parameters of Vlp structural diversity and expression that distinguish the Vlp system from previously described examples of antigenic variation. VlpA, VlpB, and VlpC are prominent amphiphilic membrane lipoproteins characterized by detergent-phase fractionation and metabolic labeling with [35S]cysteine and [3H]palmitate. VlpA is distinguished from VlpB and VlpC by its selective labeling with [35S]methionine; VlpB and VlpC are distinguished by specific epitopes defined by surface-binding monoclonal antibodies (MAbs); a third MAb defines a surface epitope shared by VlpB and VlpC (but absent from VlpA). Each Vlp displays 12 to 30 spontaneous size variant forms comprising a periodic ladder that could also be generated by partial trypsin digestion of individual Vlp size variants. Different periodic intervals within VlpB and VlpC further distinguish these two products structurally. Mycoplasma colony opacity correlates inversely with Vlp size. Each Vlp undergoes independent, oscillating high-frequency phase variation in isogenic populations and can be expressed individually or concomitantly with other Vlps in a noncoordinate manner. All seven possible combinations of these three products were observed; however, no variants were found that lacked a Vlp. High-frequency size variation of each Vlp superimposed on combinatorial diversity in Vlp expression yields greater than 10(4) possible structurally distinct Vlp mosaics, of which 104 were documented along with 24 of 42 possible transitions among the seven Vlp combinations. In addition to these features, VlpA, VlpB, and VlpC were specifically recognized by serum antibodies from swine with experimental M. hyorhinis SK76-induced arthritis, indicating expression and immunogenicity of Vlps in the natural host. The structure and variation of Vlps and their known involvement in MAb-mediated modulation of mycoplasma-infected host cell properties and mycoplasma killing are discussed in relation to the surface architecture and adaptive potential of the wall-less mycoplasmas.

Animals↗