Mechanism Of Antigenic Variation Research Articles

Mechanisms of Antigenic Variation in Borrelia hermsii and African Trypanosomes

Mechanisms of Antigenic Variation in Borrelia hermsii and African Trypanosomes

Activation of a vmp pseudogene in Borrella hermsii: an alternate mechanism of antigenic variation during relapsing fever

The relapsing fever agent, Borrelia hermsii, undergoes multiphasic antigenic variation to evade its host's immune response. A frequently observed switch is serotype 7 to 26. Unlike silent vmp genes previously characterized, the transcriptionally silent vmp26 sequence was a pseudogene in lacking a start codon. In serotype 7 the location of the silent vmp26 sequence just downstream of vmp7 on the expression plasmid, as well as on the silent plasmid, was also unique. The demonstration of a predicted circular recombination product in serotype 7 but not serotype 21 populations indicates that the pseudogene was activated by an intramolecular recombination producing a deletion of DNA between 20-nucleotide direct repeats in vmp7 and psi vmp26.

Genetic mechanisms of antigenic variation in infectious bursal disease virus: analysis of a naturally occurring variant virus.

The major immunogenic protein VP2 from a pathogenic field isolate (variant A virus) of infectious bursal disease virus (IBDV) was cloned and sequenced to examine antigenic variations. The VP2 open reading frame consists of 1509 nucleotides and codes for a 503 amino acid protein. Overall, the VP2 amino acid sequence of the variant A virus shares 98.6% identity with VP2 genes from other published IBDV strains. However, within the central region of VP2 (amino acids 222-334) lies a highly divergent area that we have termed the variable domain. Relative to five other IBDV isolates, a total of six amino acid changes occur within the variable domain of the variant A virus. At positions 284-288, a substitution of isoleucine to threonine, a decrease in the number of Chou and Fasman beta turns, and a switch from a hydrophilic to a hydrophobic region are found only in the variant A virus. Together these changes predict a decrease in antigenicity as determined by calculation of potential antigenic sites. This suggests that only minor changes within VP2 contributed to the emergence of a variant virus that can cause disease in immunized birds.

Molecular mechanisms of antigenic variation in Neisseria gonorrhoeae.

Neisseria gonorrhoeae is the causative agent of the sexually transmitted disease gonorrhoea. Gonorrhoea has been recorded in man throughout the medieval and modern eras and possibly since biblical times (Cheng, 1988). This long association between N. gonorrhoeae and humans can be attributed to:(1) an efficient mode of transmission; (2) a high percentage of asymptomatic carriers; (3) the rare occurrence of lethality, and (4) the ability of the bacterium to avoid an immune response. This Gram-negative diplococcus (the gonococcus) does not colonize any other ecological niche besides the human body. A consequence of this limited host range is a lack of a valid animal model. Therefore, a majority of facts concerning gonococcal pathogenesis result from in vitro studies, supported by information gained by occasional experiments in human volunteers. The focus of this chapter is to examine mechanisms used by the bacterium to evade the immune response. Selected topics on other aspects of gonococcal pathogenesis will be presented but for wider analysis the reader is referred to review articles about gonorrhoea by Cannon and Sparling (1984) or Brooks and Donegan (1985) and specifically about antigenic variation by Seifert and So (1988), Swanson and Koomey (1989), or Meyer et. al. (1990).

Mechanism of antigenic variation in an individual epitope on influenza virus N9 neuraminidase

Monoclonal antibodies which inhibit influenza virus neuraminidase (NA) and which therefore indirectly neutralize virus infectivity bind to epitopes located on the rim of the active-site crater. The three-dimensional structure of one of these epitopes, recognized by monoclonal antibody NC41, has previously been determined (W. R. Tulip, J. N. Varghese, R. G. Webster, G. M. Air, W. G. Laver, and P. M. Colman, Cold Spring Harbor Symp. Quant. Biol. 54:257-263, 1989). Nineteen escape mutants of influenza virus A/tern/Australia/G70c/75 (N9) NA selected with NC41 were sequenced. A surprising restriction was seen in the sequence changes involved. Ten mutants had a Ser-to-Phe change at amino acid 372, and six others had mutations at position 367. No escape mutants with changes at 369 or 370 were found, although these mutations were selected with other antibodies and rendered the epitope unrecognizable by antibody NC41. Another N9 NA, from A/ruddy turnstone/NJ/85, which differs by 14 amino acids from the tern virus NA, still bound antibody NC41. Epitope mapping by selecting multiple escape mutants with antibody NC41 thus identified only three of the five polypeptide loops on NA that contact the antibody. Escape mutants selected sequentially with three different monoclonal antibodies showed three sequence changes in two loops of the NC41 epitope. The multiple mutants were indistinguishable from wild-type virus by using polyclonal rabbit antiserum in double immunodiffusion tests, but NA inhibition titers were fourfold lower. The results suggest that although the NC41 epitope contains 22 amino acids, only a few of these are so critical to the interaction with antibody that a single sequence change allows selection of an escape mutant. In that case, the variety of amino acid sequence changes which can lead to polyclonal selection of new epidemic viruses during antigenic drift might be very limited.

Structural and serological evidence for a novel mechanism of antigenic variation in foot-and-mouth disease virus

Changes resulting in altered antigenic properties of viruses nearly always occur on their surface and have been attributed to the substitution of residues directly involved in binding antibody. To investigate the mechanism of antigenic variation in foot-and-mouth disease virus (FMDV), variants that escape neutralization by a monoclonal antibody have been compared crystallographically and serologically with parental virus. FMDVs form one of the four genera of the Picornaviridae. The unenveloped icosahedral shell comprises 60 copies each of four structural proteins VP1-4. Representatives from each of the genera have similar overall structure, but differences in the external features. For example, human rhinovirus has a pronounced 'canyon' that is proposed to contain the cell attachment site, whereas elements of the attachment site for FMDV, which involves the G-H loop (residues 134-160) and C-terminus (200-213) of VP1, are exposed on the surface. Moreover, this G-H loop, which is a major antigenic site of FMDV, forms a prominent, highly accessible protrusion, a feature not seen in other picornaviruses. It is this loop that is perturbed in the variant viruses that we have studied. The amino acid mutations characterizing the variants are not at positions directly involved in antibody binding, but result in far-reaching perturbations of the surface structure of the virus. Thus, this virus seems to use a novel escape mechanism whereby an induced conformational change in a major antigenic loop destroys the integrity of the epitope.

Vesicular stomatitis new jersey virus glycoprotein gene sequence and neutralizing epitope stability in an enzootic focus

Vesicular stomatitis New Jersey (VS NJ) virus is capable of undergoing rapid evolution in nature and therefore has the potential for antigenic variation. We selected an area of Costa Rica where VS NJ virus is enzootic to study whether this virus used the mechanism of antigenic variation to persist in nature. Three sentinel herds and three nonsentinel herds were observed from 1986 to 1988. Eleven VS N1 virus isolates were collected from naturally infected cattle. Remarkably, nine animals that were bled prior to reinfection with VS NJ virus had neutralizing antibody titers up to 1: 102,400 yet virus was isolated from, and disease was observed in, these animals. Sequence analysis of the portion of the glycoprotein gene coding for the neutralizing epitopes demonstrated that all virus isolates were 98–100% similar with no indication of specific genetic variation. The 3′ end of the glycoprotein gene also remained stable in that all isolates were again 98–100% similar in nucleotide sequence. Each isolate was neutralized to equivalent titers with monoclonal antibodies directed against four neutralizing epitopes on the glycoprotein. Additionally, preisolation sera from each animal were able to neutralize the virus that caused the subsequent disease. These results provide evidence that antigenic variation is not a mechanism used by VS NJ virus to persist in an enzootic focus of Costa Rica.

A novel telomeric gene conversion in Trypanosoma brucei

Gene conversion is one mechanism of antigenic variation in Trypanosoma brucei. Variant surface glycoprotein (VSG) genes are duplicated by this process to telomeric locations from which they may be expressed. We examined four independent antigenic switches in which the IsTaR 1.1 minichromosomal VSG gene is duplicated to a large chromosome where it is expressed. An unusual feature of three of these telomeric gene conversions is that the distance between the VSG gene and the end of the chromosome is identical for both the basic and duplicated copies following the antigenic switch. This suggests that the gene conversion is initiated 5′ to the VSG gene and extends to the end of the telomere. The data also suggest that events other than simple nucleotide addition account for telomeric growth.

Recombination among Protein II genes of Neisseria gonorrhoeae generates new coding sequences and increases structural variability in the Protein II family

Expression of Neisseria gonorrhoeae Protein II (P.II) is subject to phase variation and antigenic variation. The P.II proteins made by one strain possess both unique and conserved antigenic determinants. To study the mechanism of antigenic variation, we cloned several P.II genes, using as probes a panel of monoclonal antibodies (MAbs) specific for unique determinants. The DNA sequences of three P.II genes showed that they shared a conserved framework, with two short hypervariable (HV) regions being responsible for most of the differences among them. We demonstrated that unique epitopes recognized by the MAbs were at least partially encoded by one of the HV regions. Moreover, we found that reassortment of the two HV regions among P.II genes occurs, generating increased structural and antigenic variability in the P.II protein family.

Surface antigens of Paramecium primaurelia: Membrane-bound and soluble forms

The surface antigens of Paramecium constitute a family of high molecular weight (ca 300 kD) iso-proteins whose alternative expression, adjusted to environmental conditions, involves both intergenic and interallelic exclusion. Since the surface antigen molecules had previously been shown to play a key role in the control of their own expression, it seemed important to compare the structural particularities of different surface antigens: the G and D antigens of P. primaurelia expressed at different temperatures, and which are coded by two unlinked loci. Here we demonstrate that in all cases a given surface antigen presents two biochemically distinct basic forms: a soluble form recovered from ethanolic extraction of whole cells, and a membrane-bound form recovered from ciliary membranes solubilized by detergent. The membrane-bound form differs from the soluble one by its mobility on SDS gels and by an electrophoretic mobility shift in the presence of anionic or cationic detergents. Furthermore, two 40–45 kD polypeptides sharing common determinants with soluble antigens were found exclusively in ethanolic extracts but not in ciliary membranes: the cross-reactivity of these light polypeptides with ethanol-extracted antigens could be demonstrated only after β-mercaptoethanol treatment. Immunological comparisons between allelic and non-allelic soluble antigens demonstrate that allelic antigens share a great number of surface epitopes, most of which are not accessible in vivo, while non-allelic antigens appear to share essentially sequence-antigenic determinants. The significance of these results is discussed in relation to the mechanism of antigenic variation.

Telomeric reciprocal recombination as a possible mechanism for antigenic variation in trypanosomes

In African trypanosomes, antigenic variation is achieved through differential gene activation, with one antigen gene being expressed at a time among a large collection of antigen-specific sequences. Transcription of the antigen gene always takes place in a telomere, but different telomeres can alternatively act as the expression site. Telomeric antigen genes can be expressed without apparent DNA rearrangement, but they can also, like non-telomeric genes, have access to the telomeric expression site through a duplicative transposition mechanism resembling gene conversion. We report here that, as previously suggested, telomeric genes may use another route to be activated. This mechanism of gene activation is by reciprocal crossing-over upstream from the gene, in the so-called 'barren' region. This allows the antigen gene to be placed in the previously activated telomere, while inactivating the formerly expressed gene by recombination into a silent environment. At least for the telomeric antigen gene described here, three possible activation mechanisms coexist.

Gene conversion as a mechanism for antigenic variation in Trypanosomes

Expression of the gene coding for the trypanosome AnTat 1.1 surface antigen is linked to the duplicative transposition of a basic copy (BC) of this gene to an expression site. In two trypanosome clones successively derived from AnTat 1.1 (AnTat 1.10 and AnTat 1.1B) we found evidence that gene conversions are involved in the transformation of the AnTat 1.1 transposed element into the two new surface antigen coding sequences. Although the three resultant mRNAs—AnTat 1.1, 1.10, and 1.1B—are different, they still share large homologies. Two of them, AnTat 1.1 and 1.1B, code for surface coats that are indistinguishable by conventional serological techniques, whereas AnTat 1.10 has been found different by the same methods. The three genomic rearrangements involve two of the five members of the AnTat 1.1 gene family. These two members are both located in unstable telomeric regions similar to the expression site, each in a different orientation with respect to the DNA terminus. We have concluded that the duplicative transposition is achieved by a gene conversion that may affect variable lengths of the same silent genes, and that different members of the same surface antigen gene family can contribute to the diversification of the antigen repertoire.

Antigenic variation during cellular aging in Paramecium tetraurelia

Clones of Paramecium tetraurelia undergoing cellular aging were exposed to two antisera, anti-32 and anti-25, raised against surface antigen isolated from cells grown at 32°C or 25°C. When young clones were exposed to the two antisera about 50% were immobilized by anti-32 while others were not immobilized by either antisera. These clones were grouped as group A or B depending upon their sensitivity or nonsensitivity to the two antisera. Young cells of group A were immobilized by anti-32 and during aging these cells either maintained the same antigenic type or transformed to another. Young group B clones were not immobilized by either antisera but gradually became sensitive to anti-32. Some clones from both groups showed sensitivity to both antisera. The possible mechanism of antigenic variation during fission age is discussed.

Surface alterations of erythrocytes in Plasmodium falciparum malaria. Antigenic variation, antigenic diversity, and the role of the spleen.

The surface of erythrocytes infected with late developmental stages of Plasmodium falciparum is profoundly altered and new antigenic determinants can be detected by surface immunofluorescence using immune squirrel monkey serum. The expression of these parasite-specific antigenic determinants on the surface of the host erythrocyte can be modulated by the presence or absence of the spleen and by immune pressure. An antigenic switch occurred when a cloned population of the Ugandan Palo Alto strain of P. falciparum was transferred from a splenectomized into an intact monkey and this switch was reversible. In another strain (Indochina-1), we showed that the parasites isolated during secondary and recrudescent peaks expressed erythrocyte-associated surface antigens different from the parasites isolated during the primary infection; six variant antigenic types distinct from the original population were isolated in this way. The passive transfer of immune serum can induce antigenic variation and this can occur in a cloned parasite. The various mechanisms of antigenic variation in P. falciparum are discussed in the context of strain-specific diversity and the role of antigenic diversity in acquired immunity.

Absence of a surface coat from metacyclic Trypanosoma v/vax: possible implications for vaccination against vivax trypanosomiasis

Trypomastigotes attached to the wall of the hypopharynx in tsetse flies infected with Trypanosoma vivax are believed to represent the true metacyclic stage of this trypanosome. Electron microscopy demonstrates that attachment is mediated by hemidesmosome-like junctions along the flagellar membrane and that none of the trypomastigotes, either attached or free in the hypopharynx lumen, possesses a surface coat comparable with that on the metacyclics of T. brucei and T. congolense and on the bloodstream stages of all salivarian trypanosomes. As the variable antigen of bloodstream and metacyclic T. brucei is located in the surface coat, the absence of the coat from metacyclic T. vivax suggests that the mechanism of antigenic variation in this species may be somewhat different from that of antigenic variation in T. brucei, and that vaccination of cattle against T. vivax may prove a simpler proposition than vaccination against T. brucei.

The genes for variant antigens in trypanosomes.

We have studied the mechanism of antigenic variation by using DNA complementary to the messenger RNAs for four variant surface glycoproteins of Trypanosoma brucei. Pure complementary DNAs were obtained by cloning as recombinant DNA in Escherichia coli. Using these complementary DNAs as hybridization probes, we have analyzed the genes for these variant surface glycoproteins. The results provide new information on the origin and evolution of antigenic variation, and on the mechanism involved in switching from one antigenic type to another.

Crossreacting determinants in variant-specific surface antigens of African trypanosomes.

A number of variant-specific surface antigens (VSSAs) were purified from clones of Trypanosoma brucei and T. congolense and tested for immunological crossreactivity. Anti-VSSA sera were clone-specific when tested by indirect immunofluorescence of living trypanosomes, but they were not clone-specific when tested by radioimmunoassay with purified 125I-labeled VSSAs. In this double-antibody radioimmunoassay every VSSA tested was precipitated by the homologous and all heterologous anti-VSSA sera. Any unlabeled VSSA could inhibit the heterologous precipitation reactions by 100%. The homologous precipitation reactions were effectively inhibited only by unlabeled homologous VSSA. Crossreactions between different VSSA molecules were also revealed by microcomplement fixation tests. The results confirm the presence, in VSSAs, of variable determinants specific to individual VSSAs and also show crossreacting determinants in all VSSAs tested, including those isolated from different species of trypanosomes. These results contrast with previous studies which failed to find evidence for immunological crossreactivity between different VSSA molecules. We suggest that the crossreacting determinants in VSSAs may represent common structural regions. The existence of such regions would have considerable implication for the development of theories and experiments concerning the mechanism of antigenic variation in African trypanosomes.

Characterisation of the surface coat of Trypanosoma congolense

THE protozoan parasites Trypanosoma congolense, T. brucei and T. vivax cause extensive disease problems to livestock in Africa1. In many cases trypanosomiasis results from mixed infections due to all three species2. Immunisation has not been successful because of the ability of the parasites to evade the host immune response by changing the antigenic structure of the surface coat3. Chemical and immuno-chemical characterisation of the surface coat of all three species should give insight into the mechanism of antigenic variation. Recent work on the surface coat of T. brucei demonstrates that antigenic variation is the result of a change in structure of a single, variant-specific, surface glycoprotein4,5. No comparable information is available on the structure of the surface coat of T. congolense and T. vivax. We report here some properties of the surface coat of T. congolense.

The Nature of Serological Relationships Among Influenza Viruses

This chapter describes some of the recent work with antigens extracted from virus-infected tissues and suspensions of influenza virus particles. In many ways these studies represent promising approaches to several problems in virology. Primary emphasis has been on serologic studies with the antigenic complexes represented by populations of virus particles. A major portion concerns antigenic relationships among strains as defined by several methods of analysis with animal antisera. An effort has been made to summarize pertinent data resulting from studies on mechanisms of antigenic variation—namely, immunologic selection and genetic exchanges. Observations of immune responses in man are also discussed. It is shown that conclusions derived from results of vaccine trials and sero-epidemiological research has complemented interpretations of analyses of virus strain antigenic composition. Modern concepts and doctrines concerning the importance and nature of antigenic relationships among strains are also reviewed and discussed in relation to application in prophylaxis of influenza by vaccination.