African trypanosomes are early divergent protozoan parasites responsible for high mortality and morbidity as well as a great economic burden among the world's poorest populations. Trypanosomes undergo antigenic variation in their mammalian hosts, a highly sophisticated immune evasion mechanism. Their nuclear organization and mechanisms for gene expression control present several conventional features but also a number of striking differences to the mammalian counterparts. Some of these unorthodox characteristics, such as lack of controlled transcription initiation or enhancer sequences, render their monogenic antigen transcription, which is critical for successful antigenic variation, even more enigmatic. Recent technological developments have advanced our understanding of nuclear organization and gene expression control in trypanosomes, opening novel research avenues. This review is focused on Trypanosoma brucei nuclear organization and how it impacts gene expression, with an emphasis on antigen expression. It highlights several dedicated sub-nuclear bodies that compartmentalize specific functions, whilst outlining similarities and differences to more complex eukaryotes. Notably, understanding the mechanisms underpinning antigen as well as general gene expression control is of great importance, as it might help designing effective control strategies against these organisms.

Plasmodium knowlesi has risen in importance as a zoonotic parasite that has been causing regular episodes of malaria throughout South East Asia. The P. knowlesi genome sequence generated in 2008 highlighted and confirmed many similarities and differences in Plasmodium species, including a global view of several multigene families, such as the large SICAvar multigene family encoding the variant antigens known as the schizont-infected cell agglutination proteins. However, repetitive DNA sequences are the bane of any genome project, and this and other Plasmodium genome projects have not been immune to the gaps, rearrangements and other pitfalls created by these genomic features. Today, long-read PacBio and chromatin conformation technologies are overcoming such obstacles. Here, based on the use of these technologies, we present a highly refined de novo P. knowlesi genome sequence of the Pk1(A+) clone. This sequence and annotation, referred to as the ‘MaHPIC Pk genome sequence’, includes manual annotation of the SICAvar gene family with 136 full-length members categorized as type I or II. This sequence provides a framework that will permit a better understanding of the SICAvar repertoire, selective pressures acting on this gene family and mechanisms of antigenic variation in this species and other pathogens.

African trypanosomes have been around for more than 100 million years, and have adapted to survival in a very wide host range. While various indigenous African mammalian host species display a tolerant phenotype towards this parasitic infection, and hence serve as perpetual reservoirs, many commercially important livestock species are highly disease susceptible. When considering humans, they too display a highly sensitive disease progression phenotype for infections with Trypanosoma brucei rhodesiense or Trypanosoma brucei gambiense, while being intrinsically resistant to infections with other trypanosome species. As extracellular trypanosomes proliferate and live freely in the bloodstream and lymphatics, they are constantly exposed to the immune system. Due to co-evolution, this environment however no longer poses a hostile threat, but has become the niche environment where trypanosomes thrive and obligatory await transmission through the bites of tsetse flies or other haematophagic vectors, ideally without causing severe side infection-associated pathology to their host. Hence, African trypanosomes have acquired various mechanisms to manipulate and control the host immune response, evading effective elimination. Despite the extensive research into trypanosomosis over the past 40 years, many aspects of the anti-parasite immune response remain to be solved and no vaccine is currently available. Here we review the recent work on the different escape mechanisms employed by African Trypanosomes to ensure infection chronicity and transmission potential.

The Apicomplexa is a phylum of parasitic protozoa, which includes the malaria parasite Plasmodium, amongst other species that can devastate human and animal health. The past decade has seen the release of genome sequences for many of the most important apicomplexan species, providing an excellent basis for improving our understanding of their biology. One of the key features of each genome is a unique set of large, variant gene families. Although closely related species share the same families, even different types of malaria parasite have distinct families. In some species they tend to be found at the ends of chromosomes, which may facilitate aspects of gene expression regulation and generation of sequence diversity. In others they are scattered apparently randomly across chromosomes. For some families there is evidence they are involved in antigenic variation, immune regulation and immune evasion. For others there are no known functions. Even where function is unknown these families are most often predicted to be exposed to the host, contain much sequence diversity and evolve rapidly. Based on these properties it is clear that they are at the forefront of host–parasite interactions. In this review I compare and contrast the genomic context, gene structure, gene expression, protein localization and function of these families across different species.

The immune evasion gene family of malaria parasites encodes variant surface proteins that are expressed at the surface of infected erythrocytes and help the parasite in evading the host immune response by means of antigenic variation. The identification of Plasmodium vivax vir orthologous immune evasion gene family from primate malaria parasites would provide new insight into the evolution of virulence and pathogenesis. Three vir subfamilies viz. vir-B, vir-D and vir-G were successfully PCR amplified from primate malaria parasites, cloned and sequenced. DNA sequence analysis confirmed orthologues of vir-D subfamily in Plasmodium cynomolgi, Plasmodium simium, Plasmodium simiovale and Plasmodium fieldi. The identified vir-D orthologues are 1–9 distinct members of the immune evasion gene family which have 68–83% sequence identity with vir-D and 71·2–98·5% sequence identity within the members identified from primate malaria parasites. The absence of other vir subfamilies among primate malaria parasites reflects the limitations in the experimental approach. This study clearly identified the presence of vir-D like sequences in four species of Plasmodium infecting primates that would be useful in understanding the evolution of virulence in malaria parasites.

Trypanosoma brucei is exposed to the adaptive immune system and complement in the blood of its mammalian hosts. The aim of this review is to analyse the role and regulation of the proteins present on the external face of the plasma membrane in the long-term persistence of an infection and transmission. In particular, the following are addressed: (1) antigenic variation of the variant surface glycoprotein (VSG), (2) the formation of an effective VSG barrier shielding invariant surface proteins, and (3) the rapid uptake of VSG antibody complexes combined with degradation of the immunoglobulin and recycling of the VSG.

Haemoflagellates of the genus Trypanosoma are prevalent in freshwater fishes and are transmitted by leeches as vectors. As demonstrated by sequence comparisons of nuclear small subunit rRNA genes, trypanosomes isolated from several fish species at different localities can be divided into at least 2 closely related types, designated Type A and Type B. A clone derived from a Type A isolate from carp (Cyprinus carpio) was used to study the anti-parasite immune response in specified pathogen-free outbred carp. Infection leads to an initial rise in parasitaemia in the blood followed by a sharp decline in all fish (acute phase). Thereafter, in some carp, parasites become undetectable both in the blood and in internal organs while, in others, low numbers can be found in the blood for up to 1 year (chronic phase). Fish that have controlled an acute infection with the clone are not only protected against an homologous challenge infection, but also against the infection with parasite lines derived from carp in the chronic phase of infection. Passive immunization experiments with IgM purified from serum of recovered carp indicate that the infection is controlled by antibodies. The anti-parasite antibody level in recovered carp remains high for many months although the parasitaemia is controlled at very low levels and the half life of IgM, t1/2=22·5 days, is comparatively short. The effective control of trypanosomes in laboratory infections is in contrast to the high prevalence in natural and farmed freshwater fish populations.

Cell surface molecules have received intense attention in recent years because of the central roles they play at the interface between the external environment and the cellular interior. Their functions include adhesion to other cells or extracellular matrices, protection against hostile physical, chemical and biological agents and the transport of metabolites into and out of the cell. In addition, cell surface molecules transduce signals across the cell membrane, relaying information inwards and presenting altered characteristics to the exterior as the environment changes.

The metacyclic form of African trypanosomes is the first to express genes for the Variant Surface Glycoprotein (VSG) and it uses an unusually predictable subset of the VSG gene repertoire. We have developed a model system for the analysis of metacyclic VSG (M-VSG) gene expression and have used this to demonstrate that, for two M-VSG genes, different modes of expression operate in the insect and mammalian phases of the life-cycle. In metacyclic-derived clones, these genes are expressed in situ, whereas they are routinely activated by duplication in bloodstream trypanosomes. The expression loci for both M-VSG genes studied are structurally simple and we present a model, based on this, for the maintenance of a separate M-VSG repertoire and expression system.

Rates at which Trypanosoma brucei rhodesiense trypanosomes switch from expression of one variable antigen type (VAT) to that of another have been determined in cloned populations that have been recently tsetse-fly transmitted. Switching rates have been determined between several, specific pairs of VATs in each population. High rates of switching were observed in 2 cloned trypanosome lines, each derived from a separate cyclical transmission of the same parental stock and each expressing a different major VAT. Five estimates of the switching rate between one particular pair of VATs were consistently high (approximately 1 × 103 switches/cell/generation). These high switching rates were similar both in bloodstream populations of mice and in populations confined to subcutaneously implanted growth chambers in mice, thus indicating that the interaction of the bloodstream population with other trypanosome populations in the lymphatics or extravascular sites in systemic infections did not influence the estimates of the rate of switching. Fourteen estimates were made of VAT-specific switching rates in bloodstream infections involving 8 combinations from among 6 VATs. Switching rate estimates were VAT-specific and showed considerable variation between different combinations of VATs — from 1.9 × 10−6 to 6.9 × 10−3 switches/cel/generation. The rates of switching to different metacyclic-VATs were, however, very similar. Summation of between 3 and 5 VAT-specific switching rate values in each of 4 experiments conducted in bloodstream infections has provided minimum estimates of the overall rate of antigenic variation: 2.0−9.3 × 10−3 switches/cell/generation. These values are between 20 and 66000-fold higher than previously published estimates. It is likely that at least 1 in every 100 trypanosomes switches its VAT expression every generation.

The inheritance of variant surface antigens in Trypanosoma brucei has been determined by identifying variable antigen types (VATs) in each of two cloned parental stocks and then examining the presence and abundance of these VATs in hybrid progeny produced when these stocks undergo genetic exchange during co-transmission through tsetse flies. Nine VATs have been identified from the repertoire of the parental stock STIB 247L and 5 VATs have been identified from the parental stock STIB 386AA; the identified VATs were exclusive to each stock. Their inheritance was elucidated using two assays. In the first, repertoire antisera (RAS) containing antibody specificities to many different VATs were raised in rabbits to the 2 parental stocks and 6 progeny clones. The presence of VAT-specific antibodies in these RAS was then determined by antibody-dependent complement-mediated lysis. In the second assay, the 2 parental stocks and 4 hybrid progeny clones were each independently transmitted through tsetse flies and VATs observed using VAT-specific antisera in indirect immunofluorescence of metacyclic trypanosomes and in bloodstream forms of fly-bitten mice. The results from both assays showed that (1) both metacyclic- and bloodstream-VATs were inherited into the progeny, (2) each hybrid progeny clone contained some VATs from both parents, (3) hybrids did not express all the VATs from either parent, (4) there was little apparent pattern as to which VATs had been inherited and which had not and (5) the VAT repertoires of the hybrid progeny appeared to be larger than those of the parents. In addition, two results indicated that control of VAT expression remains unaltered after genetic exchange. Firstly, the immunofluorescence results showed that VATs present in hybrid trypanosomes were expressed at the same stage during an infection and at approximately the same prevalence as in the parent. Secondly, a double-labelling experiment using direct immunofluorescence indicated that individual hybrid trypanosomes did not generally simultaneously express more than one VAT. Taken together, these results demonstrate that recombinant VAT repertoires are created when trypanosomes undergo genetic exchange and that genetic exchange is a mechanism whereby the generation of new serodemes can occur.

The paper describes the development and analysis of a mathematical framework for the study of the within-host population dynamics of the interaction between macroparasites and the human immune system. Simple models of this interaction based on the proliferation of T cell clones specific to parasite antigen, and the impact of clonal expansion on parasite survival, capture the basic features of age-related changes in worm loads within human communities. The model is generalized to multiple epitopes on a single antigen, and reveals competitive exclusion amongst T cells, with a single clone becoming immunodominant in the absence of cross-reactive responses and genetic variation. The introduction of genetic heterogeneity and concomitant variability in the immunogenicity of specific epitopes induces additional complexity into the dynamical interaction. Most importantly, multiple epitope models with antigenic variation suggest that the immunodominant response may not necessarily be targeted at the epitope at which some strains show the greatest immunogenicity. High immunogenicity at a particular epitope can be masked by genetic variability even though many of the variants are more immunogenic at this epitope by comparison with the epitope to which the immunodominant immunological response is directed.

Antigenic variation of the intestinal protozoan parasite Giardia lamblia is caused by an exchange of the parasite's variant surface protein (VSP) coat. Many investigations on antigenic variation were performed with G. lamblia clone GS/M-83-H7 which produces surface antigen VSP H7. To generate novel information on giardial vsp gene transcription, vsp RNA levels were assessed by quantitative reverse transcription-(RT)-PCR in both axenic VSP H7-type trophozoites and subvariants obtained after negative selection of GS/M-83-H7 trophozoites by treatment with a cytotoxic, VSP H7-specific monoclonal antibody. Our investigation was not restricted to the assessment of the sense vsp transcript levels but also included an approach aimed at the detection of complementary antisense vsp transcripts within the two trophozoite populations. We found that sense vsp H7 RNA predominated in VSP H7-type trophozoites while sense RNA from only one (vsp IVg) of 8 subvariant vsp genes totally analysed predominated in subvariant-type trophozoites. Interestingly, the two trophozoite populations exhibited a similar relative distribution regarding the vsp H7 and vsp IVg antisense RNA molecules. An analogous sense versus antisense RNA pattern was also observed when the transcripts of gene cwp 1 (encoding cyst wall protein 1) were investigated. Here, both types of RNA molecules only appeared after cwp 1 had been induced through in vitro encystation of the parasite. These findings for the first time demonstrated that giardial antisense RNA production did not occur in a constitutive manner but was directly linked to complementary sense RNA production after activation of the respective gene systems.

Intra-clonal phenotypic (antigenic) variation is used by many pathogens to evade the consequences of immune-mediated killing by mammalian hosts. In this substantially theoretical article, I emphasise that antigenic variation (sensu stricto) involves no change in genotype; its importance as a mechanism for promoting pathogen transmission and its polyphyletic origin. From a functional perspective, antigenic variation is constrained by the requirement to meet five conditions. These are: capability to express several antigens against which functional immunity predominates; capability to interact with the environment; mutually exclusive expression of variable antigens in each cell within an infection; mutually exclusive expression in the within-host pathogen population and the capability for population growth within a host. Meeting these conditions leads to chronicity of infection and high rates of hierarchical and reversible switching of expression between variable antigens. The organisation of hierarchical expression is discussed in some detail.

There are strong biological, evolutionary and immunological arguments for predicting extensive polymorphism among helminth parasites, but relatively little data and few instances from which the selective forces acting on parasite diversity can be discerned. The paucity of information on intraspecific variation stands in contrast to the fine detail with which helminth species have been delineated by morphological techniques, accentuating a trend towards considering laboratory strains as representative of a relatively invariant organism. However, in the fast-moving evolutionary race between host and parasite one would predict a monomorphic species would be driven to extinction. We review the arena of intraspecific variation for the major helminth parasites, ranging from biological properties such as host or vector preference, to biochemical and immunological characteristics, as well as molecular markers such as DNA sequence variants. These data are summarized, before focusing in more detail on polymorphisms within protein-coding genes of potential relevance to the host-parasite relationship, such as vaccine candidates. In particular, we discuss the available data on a number of major antigens from the filarial nematode Brugia malayi. Information is currently too sparse to answer the question of whether there is antigenic variation in filariasis, but the indications are that proteins from the blood-borne microfilarial stage show significant intraspecific variability. Future work will define whether polymorphisms in these antigens may be driven by exposure to the host immune response or reflect some other facet of parasite biology.

Philasterides dicentrarchi is a histophagous ciliate causing systemic scuticociliatosis in cultured turbot. This study demonstrates that turbot which survive this disease have serum antibodies that recognize ciliary antigens of this ciliate in ELISA and immobilize/agglutinate the ciliate in vitro. Mouse sera raised against ciliary antigens and integral membrane proteins are likewise capable of immobilizing/agglutinating the ciliates, indicating that P. dicentrarchi, like other ciliates, expresses surface immobilization antigens. Furthermore, the antigen agglutinating reaction induces the parasite to shed its surface antigens rapidly, replacing them with others with different specific serology. This antigen shedding and variation response is similar to that detected in other protozoan parasites. Immunization of turbot with ciliate lysate plus adjuvant or with formalin-fixed ciliates induced synthesis of agglutinating antibodies and conferred a degree of protection against challenge infection, suggesting that the response to surface antigens may play an important role in defence against this pathogen, SDS–PAGE and immunoblotting studies indicated the existence of a predominant polypeptide of about 38 kDa in the ciliary antigen and membrane protein fractions, and this may be the principal surface antigen of P. dicentrarchi.

Intra-erythrocytic stages of malaria parasites can alter the surface of their host cells and release toxins which induce the production of cytokines, which in turn can up- or down-regulate the expression of adhesion receptors on the surface of microvascular endothelial cells. New adhesion receptors on endothelial cells provide the parasite with increased chances of survival despite an increasing level of host immunity. In order to take advantage of these new opportunities for survival, the parasite itself needs to make best use of its considerable ability to vary its surface antigens and adherent molecules. The paper describes the various players in this survival game and articulates a working hypothesis to explain how it may all fit together.

Antigenic variation is a mechanism exploited by parasitic protozoa to evade the host's immune response. Studies on such variation are of cardinal importance in approaching the practical problems of vaccine design and diagnosis of infection by serological tests. Coincidently these studies have revealed novel gene expression mechanisms which are of fundamental biological interest (Vickerman and Barry, 1982). The African (salivarian) trypanosomes all exhibit antigenic variation and the process has been studied in Trypanosoma brucei subspecies in particular.