Molecular Basis Of Antigenic Variation Research Articles

The molecular basis of antigenic variation among A(H9N2) avian influenza viruses

Avian influenza A(H9N2) viruses are an increasing threat to global poultry production and, through zoonotic infection, to human health where they are considered viruses with pandemic potential. Vaccination of poultry is a key element of disease control in endemic countries, but vaccine effectiveness is persistently challenged by the emergence of antigenic variants. Here we employed a combination of techniques to investigate the genetic basis of H9N2 antigenic variability and evaluate the role of different molecular mechanisms of immune escape. We systematically tested the influence of published H9N2 monoclonal antibody escape mutants on chicken antisera binding, determining that many have no significant effect. Substitutions introducing additional glycosylation sites were a notable exception, though these are relatively rare among circulating viruses. To identify substitutions responsible for antigenic variation in circulating viruses, we performed an integrated meta-analysis of all published H9 haemagglutinin sequences and antigenic data. We validated this statistical analysis experimentally and allocated several new residues to H9N2 antigenic sites, providing molecular markers that will help explain vaccine breakdown in the field and inform vaccine selection decisions. We find evidence for the importance of alternative mechanisms of immune escape, beyond simple modulation of epitope structure, with substitutions increasing glycosylation or receptor-binding avidity, exhibiting the largest impacts on chicken antisera binding. Of these, meta-analysis indicates avidity regulation to be more relevant to the evolution of circulating viruses, suggesting that a specific focus on avidity regulation is required to fully understand the molecular basis of immune escape by influenza, and potentially other viruses.

A Statistically Rigorous Method for Determining Antigenic Switching Networks

Many vector-borne pathogens rely on antigenic variation to prolong infections and increase their likelihood of onward transmission. This immune evasion strategy often involves mutually exclusive switching between members of gene families that encode functionally similar but antigenically different variants during the course of a single infection. Studies of different pathogens have suggested that switching between variant genes is non-random and that genes have intrinsic probabilities of being activated or silenced. These factors could create a hierarchy of gene expression with important implications for both infection dynamics and the acquisition of protective immunity. Inferring complete switching networks from gene transcription data is problematic, however, because of the high dimensionality of the system and uncertainty in the data. Here we present a statistically rigorous method for analysing temporal gene transcription data to reconstruct an underlying switching network. Using artificially generated transcription profiles together with in vitro var gene transcript data from two Plasmodium falciparum laboratory strains, we show that instead of relying on data from long-term parasite cultures, accuracy can be greatly improved by using transcription time courses of several parasite populations from the same isolate, each starting with different variant distributions. The method further provides explicit indications about the reliability of the resulting networks and can thus be used to test competing hypotheses with regards to the underlying switching pathways. Our results demonstrate that antigenic switch pathways can be determined reliably from short gene transcription profiles assessing multiple time points, even when subject to moderate levels of experimental error. This should yield important new information about switching patterns in antigenically variable organisms and might help to shed light on the molecular basis of antigenic variation.

Nuclear architecture, genome and chromatin organisation in Trypanosoma brucei

The nucleus of the human pathogen Trypanosoma brucei not only has unusual chromosomal composition, characterised by the presence of megabase, intermediate and minichromosomes, but also chromosome and gene organisation that is unique amongst eukaryotes. Here I provide an overview of current knowledge of nuclear structure, chromatin organisation and chromosome dynamics during interphase and mitosis. New technologies such as chromatin immunoprecipitation, in combination with new generation sequencing and proteomic analysis of subnuclear fractions, have led to novel insights into the organisation of the nucleus and chromatin. In particular, we are beginning to understand how universal mechanisms of chromatin modifications and nuclear position effects are deployed for parasite-specific functions and are centrally involved in genomic organisation and transcriptional regulation. These advances also have a major impact on progress in understanding the molecular basis of antigenic variation.

Single-amino-acid mutation in the HA alters the recognition of H9N2 influenza virus by a monoclonal antibody

We explored the molecular basis of antigenic variation by comparing two H9N2 subtype avian influenza viruses, A/Chicken/Shandong/6/96 (CK/SD/6) and A/Chicken/Guangxi/10/99 (CK/GX/10), that react differently to a monoclonal antibody C/B3. To assess the genetic basis for this antigenic difference, we used reverse genetics to generate a series of chimera and mutants of these two viruses. We found that a single-amino-acid substitution of asparagine for serine at position 145 (S145N) in the HA protein prevents the reaction of CK/SD/6 virus with C/B3. Substitution of serine for asparagine at the same position (N145S) enables the CK/GX/10 to react with C/B3 in hemaglutinin inhibition, immunofluorescence and neutralization assays. We further demonstrated that the amino acid N145 in the H9 HA protein is glycosylated. Our results provide experimental evidence that the glycosylation of HA oligosaccharide attachment sites implicated in antibody binding could have a role in antigenic variation.

Antigenic variation in African trypanosomes: monitoring progress.

Antigenic variation is central to the success of African trypanosomes and other eukaryotic, bacterial and viral pathogens. Our understanding of the control and execution of this immune evasion strategy in trypanosomes is incomplete, despite the molecular basis of antigenic variation being first described over 20 years ago. Recent research progress in this field is highlighted here and some of the unresolved questions raised.

Modelling Trypanosoma congolense parasitaemia patterns during the chronic phase of infection in N'Dama cattle.

We reanalyzed parasitaemia profiles of the trypanotolerant N'Dama cattle (Bos taurus), consecutively infected with the same four clones of Trypanosoma congolense. Our analysis shows that each individual parasitaemia is characterized by progressively longer intervals between parasites waves. This pattern is most visible during the chronic phase of infection. In addition, the last of the four infections had a significantly larger overall duration of inter-wave intervals. We retrieved these patterns by numerical simulations of a mathematical model, which incorporates assumptions about the molecular basis of antigenic variation and about the anti-parasitic major immune processes. Six potential factors that may determine parasitaemia pattern were studied: carrying capacity of the host environment, intrinsic growth rate of the parasite, affinity maturation of the immune response, immune cell birth and death rate, levels of antibodies to variant surface glycoprotein and levels of antibodies to invariant antigens. Our simulations suggest that the first five factors are not likely to determine the chronic phase parasitaemia pattern whereas the sixth one, namely, antibody response to invariant antigens, yielded profiles consistent with the experimental data. Being cumulative, the immune response to anti-invariant antigens may be increasingly effective as infection proceeds and in successive infections. Comparisons between N'Dama and Zebu and between chronic and acute phases will be needed to make a statement on the role of this phenomenon in trypanotolerance.

Antigenic Variation of SIV: Mutations in V4 Alter the Neutralization Profile

Antigenic variation is a characteristic feature of lentiviral infection. The SIV/macaque model of AIDS provides an ideal system in which to investigate the molecular basis of antigenic variation. The purpose of this study was to genetically map the nucleotide changes inenvthat alter the neutralization phenotype of SIV. Serum taken from an SIVmac239-infected macaque (2D) at 30 weeks postinoculation was found to neutralize the input virus (SIVmac239) and an isolate, P9, obtained at 10 weeks p.i., but did not neutralize two other isolates, P13 and P23, obtained at 20 and 52 weeks, respectively. Sequence analysis of these virus variants revealed clustered amino acid changes in V1 and single base pair changes in V2–V4 of P13 and P23. Infectious recombinant viruses in which the V1 and V1–V3 sequences of SIVmac239 were replaced with those of P13 or P23 retained the neutralization profile of SIVmac239; both were neutralized by macaque 2D serum. Recombinants containing the entire surface glycoprotein (gp120) (V1–V5) and the 5′ portion of gp41 of P13 and P23 and those containing gp120 sequences from V4 through the 5′ portion of the transmembrane glycoprotein (gp41) were not neutralized by 2D serum. Using a panel of monoclonal antibodies in radioimmunoprecipitation assays, P23 and recombinants containing V4 and V5 of P23 were shown to be antigenically distinct from P13 and SIVmac239. The majority of the amino acid changes in the antigenically distinct viruses were clustered in V4 (amino acids 413–418) and these changes created new potential N-linked glycosylation sites. This study demonstrates that a small number of specific amino acid changes (amino acids 412 to 418 in theenvgene) in the V4 region of the SIV envelope glycoprotein can alter antibody recognition and neutralization and that these phenotypic changes may be associated with altered glycosylation of the envelope.

Molecular basis of antigenic variation between the glycoproteins C of respiratory bovine herpesvirus 1 (BHV-1) and neurovirulent BHV-5

Herpesvirus glycoprotein C (gC) functions as a major virus attachment protein. The gC sequence of the neurovirulent bovine herpesvirus type 5 (BHV-5) virus was determined and compared with the gC sequence of the nonneurovirulent BHV-1. Alignment of the predicted amino acid sequences of BHV-1 and BHV-5 gC ORFs showed that the amino-terminal third of the protein differed between the two viruses. Whole or subgenomic fragments of gC coding regions from both viruses were expressed as trpE—gC fusion proteins in Escherichia coli to map linear epitopes defined by type-specific murine monoclonal antibodies (MAbs). Based on the reactivity of BHV-1-specific MAbs with the recombinant proteins, two epitopes were mapped between BHV-1 gC residues 22 and 172. Unidirectional deletion of these residues at the carboxy end mapped one within residues 22–69 and the other within residues 103–122. Two BHV-5-specific MAbs identified an epitope coding region within BHV-5 gC residues 31–78. Bovine antisera against BHV-1 and BHV-5 showed specificity to BHV-1 gC residues 22–69 and to BHV-5 gC residues 31–78, respectively, in a type-specific manner.

Molecular basis of antigenic variation in infectious bursal disease virus

Four antigenically different strains of infectious bursal disease virus (IBDV), characterized by their reactivities with a panel of neutralizing monoclonal antibodies (MAbs), were selected to determine the molecular basis of antigenic variation. The large genome segment A, encoding the structural proteins of the U.S. variants GLS, DS326, E/Del and the vaccine strain D78, was cloned and sequenced. Comparison of the deduced amino acid sequences of the U.S. variants with other IBDV strains showed that most of the amino acid substitutions occur in the central region between residues 212 to 332, especially in the two hydrophilic regions between residues 212 to 223 and residues 314 to 324 of VP2 protein. By comparing the amino acid sequences of these variant viruses and their reactivities with IBDV specific MAbs, the putative amino acids involved in the formation of virus-neutralizing epitopes were identified. Comparison of the D78 versus PBG98 sequence showed that Gln at position 249 (Gln249) appears to be critical in binding with MAb B69. Similarly, comparison of the U.S. variant sequences with other serotype 1 sequences showed unique substitution(s) at residue Glu321 in GLS, residues Ile286, Asp318, Glu323 in E/Del, and residues Glu311 and Gln320 in DS326, which could be potential residue(s) involved in the recognition of MAb57, MAb67, and MAb179 epitopes, respectively. Comparison of the serotype 1 and serotype 2 sequences revealed that serotype 2 OH strain lacks the conserved amino acid sequence motif, S-W-S-A-S-G-S, found in all virulent strains, as well as the second hydrophilic peak region, indicating a possible role of these residues in the serotype specificty or the pathogenicity of the virus. Phylogenetic analysis of the IBDV proteins indicated that the U.S. variants are antigenically different from geographically distant European viruses.

Glycosylation of the variant surface antigens of Trypanosoma brucei.

The variant-specific protein that forms the surface coat of the bloodstage trypanosome of Trypanosoma brucei is a glycoprotein, with oligosaccharides attached by covalent linkage to specific residues in the protein. The organism is able to express sequentially individual genes from a large repertoire of surface antigen genes and glycosylation of each of these gene products has been shown by binding to lectins (Strickler et al. 1978), by the direct detection of specific sugars associated with the protein (Allsopp and Njogu 1974; Johnson and Cross 1977) or by the effects of various inhibitors of protein glycosylation on the biosynthesis of the protein (Strickler and Patton 1980; Rovis and Dube 1981). The variant-specific proteins of the related trypanosomes T. congolense (Rautenberg et al. 1981; Reinwald et al. 1981) and T. equiperdum (Baltz et al. 1977) are also glycosylated. The molecular basis of antigenic variation and the biochemistry of the variant surface glycoproteins (VSGs) have been reviewed recently (Turner 1982; Borst and Cross 1982; Englund et al. 1982). This chapter will describe what is known about the location, biosynthesis, processing and structure of the carbohydrate attached to VSGs of T. brucei and will assess their possible significance for the biology of the parasite.

Sequential mutations in hemagglutinins of influenza B virus isolates: definition of antigenic domains.

Comparative analysis of the amino acid sequences of hemagglutinins (HAs) of influenza B/Lee/40, B/Md/59, and B/HK/73 viruses has allowed examination of the molecular basis of antigenic variation in type B viruses. As seen with influenza type A viruses, antigenic drift in influenza B viruses proceeds mostly through the accumulation of amino acid substitutions within the HA1 portion of the HA molecule. However, the rate of variation observed among the influenza B virus HAs appears to be significantly lower than the observed rate of variation among influenza A virus HAs. The overall rate of amino acid change in the HA1s of the influenza B viruses studied is 2% per 10 years, whereas the HA1s of H3 influenza A viruses vary by 9.2% per 10 years. The sequences of the influenza B HAs were also examined in relation to the three-dimensional model for the A/Aichi/2/68 HA. When the primary amino acid sequences are compared, it appears that most of the important structural features of the type A HAs--such as the sialic acid binding site, the disulfide linkages, and the stem structure of the trimer--are conserved in the influenza B virus HAs. Regions are also identified where extensive amino acid substitutions have occurred among the three antigenically distinct influenza B virus HAs. The locations of these areas in the B HA structure correspond to antigenic regions proposed for the A virus HAs. In addition, modulation of antigenic regions in B virus HAs may also occur through amino acid deletions and variation in glycosylation sites.

Influenza

Influenza is the last great uncontrolled plague of mankind. Pandemics and epidemics occur at regular time intervals. The influenza viruses are divided into the types A, B and C and show unique variability of their surface antigens (hemagglutinin and neuraminidase). Influenza viruses of type A show the largest degree of antigenic variation which, in turn, resulted in the definition of a number of subtypes, each comprising many strains. By comparison, influenza viruses of types B and C exhibit much less variation of their surface antigens. As a consequence, no subtypes but many different strains have been recognized. The degree of antigenic variation correlates with the epidemiologic significance of the virus types, type A being the most and type C the least important. Two different kinds of antigenic variation have been recognized: In the case of minor variation of one or both surface antigens, the term "antigenic drift" is employed. Antigenic drift occurs with all three types of virus, it is caused by point mutations which increase the chance of survival of mutants in the diseased host. In addition, influenza A viruses show sudden and complete changes of their surface antigens in regular time intervals, resulting in the appearance of new subtypes. This event is called "antigenic shift". The mechanisms responsible for antigenic shift are poorly understood, only. In addition to the recycling of preceding subtypes, reassortment resulting from double infection of cells with strains of human and animal origin are considered possible explanations. By use of modern DNA recombinant technology, the base sequences of a series of virus genes and, as a consequence, the amino acid sequence of the corresponding antigens have been determined. By means of monoclonal antibodies, the antigenic structure of many influenza antigens has been further elucidated. It can be expected that further research on the molecular basis of antigenic variation could finally result in an understanding of the causal mechanisms. It is an outstanding feature of the epidemiology of influenza A viruses that a family of related strains prevails for a certain period of time and disappears abruptly as a new subtype emerges.(ABSTRACT TRUNCATED AT 400 WORDS)

N-terminal amino acid sequences of variant-specific surface antigens from Trypanosoma brucei.

THE major pathogenic African (salivarian) trypanosomes can evade the immune response through a process of antigenic variation (reviewed in ref. 1). Mainly due to Gray's work2,3, this variation is thought to be phenotypic—involving sequential expression of alternative genes and not resulting from contemporaneous mutation. The capacity for variation which exists within a clone, isolate or species, or within strains circulating within a geographical area, is uncertain (though probably extensive) and is relevant to an evaluation of the feasibility of developing vaccines. Although serotyping of trypanosome populations has been refined through the use of fluorescence methods4,5, there is a need to characterise the relevant antigens and investigate the molecular basis of antigenic variation. Other workers6–9 identified putative variable antigens and found them to be soluble glycoproteins10–12, probably located in the surface coat13,14. The antigen preparations obtained, however, were generally heterogeneous, frequently unstable or in low yield. A recent report15 described the complete purification in good yield of individual variant-specific surface antigens (VSSAs) from a series of clones of Trypanosoma brucei. The VSSAs seem to be the primary mediators of antigenic variation in T. brucei. Each anti-genically homogeneous trypanosome clone yields one predominant glycoprotein antigen composed of a single polypeptide chain (apparent molecular weight 65,000 as determined by sodium dodecyl sulphate (SDS)–polyacrylamide gel electrophoresis) and, depending on the variant, 7–17% (w/w) carbohydrate (unpublished results of J. G. Johnson). The VSSA seems to be the major or sole constituent of the surface coat and comprises 5–10% of the total cell protein. Studies of the amino acid sequences, carbohydrate constitution and cell-surface organisation of several VSSAs are in progress, and we report here the N-terminal amino acid sequences of VSSAs isolated from four closely related variants.