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Variant genes and the spleen in Plasmodium vivax malaria
International Journal for Parasitology 34 (2004) 1547–1554 www.parasitology-online.com
Invited review
Variant genes and the spleen in Plasmodium vivax malaria Hernando A. del Portilloa,*, Michael Lanzerb, Sergio Rodriguez-Malagaa, Fidel Zavalac, Carmen Fernandez-Becerraa a
Departamento de Parasitologia, Instituto de Cieˆncias Biomedicas, Universidade de Sa˜o Paulo, Av. Lineu Prestes 1374, Sa˜o Paulo, SP 05508-900, Brazil b Hygiene-Institut, Abteilung Parasitologie, Universita¨t Heidelberg, Heidelberg, Germany c Johns Hopkins Malaria Research Institute, Johns Hopkins University, Baltimore, MD, USA Received 25 May 2004; received in revised form 18 October 2004; accepted 18 October 2004
Abstract It is generally accepted that Plasmodium vivax, the most widely distributed human malaria, does not cytoadhere in the deep capillaries of inner organs and thus this malaria parasite must have evolved splenic evasion mechanism in addition to sequestration. The spleen is a uniquely adapted lymphoid organ whose central function is the selective clearance of cell and other particles from the blood, and microbes including malaria. Splenomegaly is a hallmark of malaria and no other disease seems to exacerbate this organ as this disease does. Besides this major selective clearance function however, the spleen is also an erythropoietic organ which, under stress conditions, can be responsible for close to 40% of the RBC populations. Data obtained in experimental infections of human patients with P. vivax showed that anaemia is associated with acute and chronic infections and it has been postulated that the continued parasitemia might have been sufficient to infect and destroy most circulating reticulocytes. We review here the basis of our current knowledge of variant genes in P. vivax and the structure and function of the spleen during malaria. Based on this data, we propose that P. vivax specifically adhere to barrier cells in the human spleen allowing the parasite to escape spleen-clearance while favouring the release of merozoites in an environment where reticulocytes, the predominant, if not exclusive, host cell of P. vivax, are stored before their release into circulation to compensate for the anaemia associated with vivax malaria. q 2004 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: vir; Plasmodium vivax variant genes; Spleen; Reticulocytes; Cytoadherence; Barrier cells
1. Antigenic variation Antigenic variation refers to the capacity of many viruses, bacteria and parasites to alter their surface proteins in order to evade the immune system and establish chronic infections (Brown and Brown, 1965, Dietsch et al., 1997, Kyes et al., 2001). Malarial parasites establish chronic infections that persist for long periods of time, despite the concurrent presence of a strong immune response directed against parasite-encoded antigens exposed on the surface of the host erythrocyte (Miller et al., 1994; Kyes et al., 2001). The host’s efforts to eliminate the pathogen are counteracted by the parasite’s capability of constantly changing the * Corresponding author. Tel.: C55 11 3091 7209; fax: C55 11 3091 7417. E-mail address: [email protected] (H.A. del Portillo).
antigenic specificity of these proteins (Brown and Brown, 1965; Brown and Hills, 1974; Reeder and Brown, 1996). This cycle of parasite killing by variant specific antibodies and outgrowth of novel variants results in recurrent waves of parasitemias. As a direct result of antigenic variation, immunity to malaria develops only slowly and rarely progresses to a condition of complete protection even in highly endemic areas where re-infections leading to patent parasitemias occur frequently (Miller et al., 1994). Brown and Brown (1965) presented the first conclusive evidence in support of antigenic variation in malarial parasites. Their work relied on an observation made by Eaton in 1939 who found that erythrocytes infected with mature stages of Plasmodium knowlesi were agglutinated by sera from rhesus monkeys immune to P. knowlesi, but not by sera from normal rhesus monkeys (Eaton, 1938). Using the ability to agglutinate schizont-infected erythrocytes as
0020-7519/$30.00 q 2004 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2004.10.012
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a serological test (SICA test), Brown and Brown showed that, if a rhesus monkey was infected with a single strain of P. knowlesi and drug cured, serum collected 2 weeks later caused specific agglutination of erythrocytes infected with this parasite (Brown and Brown, 1965). When the monkey was reinfected with the same strain, however, the parasite population that was recovered was not recognised by the serum from the first convalescent blood. Experiments with clonal populations and their respective agglutinating antibodies further demonstrated the existence of antigenic variation in P. knowlesi and paved the way to the identification and characterisation of the variant antigens on the surface of infected erythrocytes (Howard et al., 1983). Antigenic variation is a regular feature of all Plasmodium species where it has been sought, including the simian malarias, P. knowlesi (Brown and Brown, 1965; Barnwell et al., 1982) and Plasmodium fragile (Handunnetti et al., 1987), the rodent malaria, Plasmodium chabaudi (McLean et al., 1982), and the human malarias, Plasmodium vivax (Mendis et al., 1988; del Portillo et al., 2001) and Plasmodium falciparum (Hommel et al., 1983; Biggs et al., 1991; Roberts et al., 1992). That all Plasmodium species apparently use antigenic variation clearly emphasises the importance of this immune evasion strategy in parasite survival, yet fails to explain why malarial parasites insert components into their host cell membranes in the first place, subsequently becoming a target for the host’s immune response. After all, the exposure of antigens on the erythrocyte looks like a suicidal action (Borst et al., 1995). It has been proposed that such deliberate parasiteprompted killing mechanism prevents fulminate parasite growth and subsequent host death, enhancing the parasite’s chances of being transmitted to another host (Saul, 1999). Increasing evidence point towards a different function of these variant antigens. When malarial parasites propagate within the erythrocyte, they alter their host cell (Aikawa, 1988). The spleen recognises abnormal erythrocytes, removes them from circulation and the erythrocyte, together with its intracellular parasite, are phagocytosed and destroyed by cells of the spleen’s reticuloendothelial system (Wyler, 1983). To avoid spleen-specific clearance the parasite needs to take action. A drastic measure is displayed by P. falciparum. This parasite, in particular its late developmental stages that cause the most damage to the host erythrocyte, avoid passage through the spleen by cytoadhesion to the endothelium of venular capillaries in the deep vascular bed of inner organs (Bignami and Bastianelli, 1889; Miller, 1969). Sequestration is mediated by the P. falciparum erythrocyte membrane protein 1 (PfEMP1), a variant antigen that renders the infected erythrocyte adhesive, allowing it to bind to a broad range of receptors on the surface of endothelial cells and uninfected erythrocytes (Baruch et al., 1995; Smith et al., 1995; Su et al., 1995). While sequestration appears to be the prime spleen evasion mechanism developed by P. falciparum, P. vivax, must have
found a different solution to avoid spleen clearance as it is widely accepted that it does not sequester in the deep vascular bed of inner organs having an obligate passage through this organ.
2. Antigenic variation in P. vivax Plasmodium vivax is the most widely distributed human malarial parasite and is estimated to cause 80–90 million cases a year and in Latin America and Asia, is the most prevalent species among the four human malarial parasites (Mendis et al., 2001). Plasmodium vivax cannot be continuously cultured in vitro, so parasites must be obtained from human patients or infected monkeys. Although this limitation has somewhat hindered research of this parasite species, it has also allowed researchers to obtain valuable information from natural parasite populations. Plasmodium vivax invades preferentially, if not exclusively, reticulocytes (Kitchen, 1938; Galinski and Barnwell, 1995). Interestingly, infected reticulocytes exhibit an ultrastructure distinct from that of P. falciparum infected erythrocytes. Whereas P. falciparum infected erythrocytes display electron-dense protrusions on their surface, called knobs, P. vivax infected reticulocytes suffer invaginations (Aikawa, 1988) and have different rheological properties that confer infected reticulocytes more deformability (Suwanarusk et al., 2004). Ultrastructural studies have identified these invaginations as caveola vesicle complexes. These structures are consistent with the Schuffner dots, small brick-red dots scattered over the host reticulocyte cytoplasm, observed in Giemsa stained thin blood smears (Schu¨ffner, 1899). The parasite induced invaginations of the host reticulocyte surface contain P. vivax-encoded antigens as demonstrated by immuno-electronmicroscopy and immunofluorescence using sera from P. vivax-infected patients (Matsamuto et al., 1988; Udagama et al., 1988). Significantly, these antigens are highly polymorphic. Thus, of 13 independent P. vivax isolates tested only some reacted with heterologous sera obtained from other P. vivax patients (Mendis et al., 1988). These results have suggested that P. vivax, like P. knowlesi, P. chabaudi and P. falciparum, use antigenic variation to establish a chronic infection. 2.1. Variant genes in P. vivax To identify gene(s) encoding variant antigens in P. vivax, we have constructed a representative genomic library of P. vivax in yeast as artificial chromosomes (YAC) and screened it for the presence of telomeric YACs (Camargo et al., 1997). Telomeric YAC clones were investigated because, in P. falciparum, subtelomeric domains contain clusters of multigene families, such as var, rif, stevor, and clag, which are implicated in antigenic variation and cytoadherence (Gardner et al., 1998; Bowman et al.,
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Fig. 1. Gene structure of virulent genes in Plasmodium falciparum, P. vivax and P. knowlesi. The two-exon (P. falciparum), three-exon (P. vivax), and 10-exon (P. knowlesi) (al-Khedery et al., 1999) structures of the var, rif, stevor, vir, and sicavar genes, are shown. Exons are depicted as boxes with introns as linking lines. Canonical signal sequences of rif and stevor genes are found in shaded boxes and predicted transmembrane domains as black boxes. Scale bars are in kilo-base pairs (kb).
1999). Several different clones were identified and one of them was completely sequenced (http://www.sanger.ac.uk/ Projects/P_vivax/). The sequence revealed the existence of a P. vivax-specific subtelomeric multigene family, termed vir (P. vivax variant genes), with ca. 600–1000 copies per haploid genome (del Portillo et al., 2001). Moreover, analysis of P. vivax chromosomes, as well as eight random telomeric YAC clones, revealed that members of this gene family are predominantly located within the subtelomeric domain of most, if not all, P. vivax chromosomes. These vir genes have a consensus three-exon structure which contrasts with the 10-exon structure of the sicavar genes of P. knowlesi and with the two-exon structure of var, rif and stevor genes of P. falciparum (Fig. 1). The 5 0 short exon lacks a predicted signal peptide sequence. The second exon is highly variant containing a predicted
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transmembrane domain and conserved cysteine residues. BLAST analysis of vir sequences from this exon predicted the existence of different subfamilies, termed A–F, varying in their extent of allele polymorphism. The third exon is of uniform size and encodes a cytosolic domain. The region covering the splice boundary between exons 2 and 3 is extremely well conserved. Similar to the P. knowlesi sicavar protein and the P. falciparum PfEMP-1 protein, typical signal sequences are lacking in vir antigens. A striking feature of the P. vivax subtelomeric domain is the lack of complex repeats. Apart from the telomeric repeat, there are no other large tandem arrays of repetitive sequence. This is in marked contrast to P. falciparum (Gardner et al., 2002) (Fig. 2). In fact a predicted gene occurs less than 1 kb from the telomeric repeat (del Portillo et al., 2001). Further hybridisation studies with a P. falciparum probe specific for subtelomeric repeats failed to cross-hybridise with nine random, non-contiguous P. vivax telomeric YAC clones, confirming that P. vivax chromosome ends lack complex repeat sequence elements (del Portillo et al., 2001). It has been postulated that complex repeat sequences facilitate ectopic recombination events between subtelomeric var genes in P. falciparum, by promoting the formation of a close, bouquet-like arrangement of ends from different chromosomes in the nucleus (Freitas-Junior et al., 2000). It is tempting to speculate that the high copy number of vir genes at chromosome ends itself promotes the pairing of, and recombination between, heterologous chromosomes, without the need of complex repeat sequence elements. Interestingly, in both P. vivax and P. falciparum, the genes involved in antigenic variation cluster within subtelomeric domains and their primary sequences are highly species specific. In contrast, internal chromosome regions appear highly conserved between P. falciparum and P. vivax (Tchavtchitch et al., 2001), consistent with the concept of a structural and functional compartmentalisation of Plasmodium chromosomes (Lanzer et al., 1993). It appears that
Fig. 2. Telomere organisation of virulent genes in Plasmodium vivax and P. falciparum. Scheme of chromosome-ends of P. falciparum and P. vivax displaying the telomere-associated repetitive DNA sequences and subtelomere-associated multigene families. Order and orientation of genes are shown. Exons are coloured boxes with introns as linking lines. (For interpretation of references to colour in this figure legend, the reader is referred to the web version of the article.)
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those genes encoding antigens located at the surface of the host cell have moved to the subtelomeric domain where they are subjected to high recombination frequencies. This in turn allows for the expansion of these genes into gene families and, at the same time, creates variants with novel antigenic phenotypes. Indeed, true multigene family orthologs of vir genes have recently been described in the subtelomeric regions of rodent and monkey malarias (Janssen et al., 2002). 2.2. Vir genes and antigenic variation Several lines of evidence initially suggested that vir genes were involved in antigenic variation in P. vivax: Firstly, analysis of expressed vir genes in natural parasite populations obtained from three different patients demonstrated that they were abundantly expressed. Furthermore, there are many different sets of variants expressed among different patients suggesting that P. vivax uses its vast repertoire of vir genes in natural parasite populations. Second, a preliminary serological survey with two different expressed vir gene tags showed that vir proteins were immunogenic and immunovariant. Third, laser confocal microscopy using fluorescently labelled affinity purified human anti-vir antibodies or anti-peptide antibodies specific for a conserved motif of vir proteins demonstrated their location at the surface of the infected reticulocyte (del Portillo et al., 2001). Together, these results established vir genes as the first virulent factor to be putatively involved in antigenic variation and chronic infections in P. vivax. To further study these vir genes, we are analysing the gene repertoire and expression of vir genes from individual patients in the Brazilian Amazon. To do so, parasite DNA and RNA are extracted from individual patients and used as templates for PCR and reverse transcriptase PCR amplifications of genomic and expressed vir sequences, respectively, using degenerate, vir-specific oligonucleotides representing subfamilies A–F. For instance, from one patient, we have generated a total of 45 different sequences (24 genomic and 21 transcribed) representing about 27 kb of vir sequences. In silico analysis of alignments made with CLUSTALX and manually edited with GeneDoc confirmed that vir genes are structured into different subfamilies varying in their extent of allele polymorphism in natural parasite populations (Fig. 3). Thus, there are highly polymorphic subfamilies such as A and C and highly conserved subfamilies such as D. Moreover, there was remarkably little overlap among all sequences clearly indicating the existence of a vast vir gene repertoire in natural infections. To determine the role of vir proteins in naturally acquired immunity we are analysing the naturally acquired humoral immune responses of P. vivax infected patients in the Brazilian Amazon using several different GST-vir tags. Results have confirmed that vir proteins are immunovariant; thus, immune sera from 23 out of 32 patients (w72%) specifically reacted against one or more of 22 GST-vir tags representing all vir subfamilies. Unexpectedly however, there was no significant difference in
Fig. 3. vir Genes are structured into conserved and highly polymorphic subfamilies in natural infections. Deduced amino acid alignments of genomic and expressed vir sequences obtained from parasites of an individual Plasmodium vivax patient were made with CLUSTALX and manually edited with GeneDoc (http://www.psc.edu/biomed/genedoc/). The unrooted tree was generated from these alignments using TreeView 32. Sequences were obtained from cloned PCR-fragments amplified with degenerate yet vir-specific subfamily oligonucleotides as described elsewhere (del Portillo et al., 2001).
the recognition of vir-tags by immune sera of first-infected or multiple-infected patients suggesting that several vir proteins are expressed on the surface of individual parasites. Laser confocal microscopy of individual infected reticulocytes using polyclonal monospecific antibodies against peptides representing different vir subfamilies have indeed proved so (Fernandez-Becerra C and del Portillo HA, unpublished). This data contrasts with similar studies in var genes of P. falciparum and with the sicavar genes of P. knowlesi (Kyes et al., 2001; Barnwell et al., 1982; Barnwell et al., 1983). Most importantly, together, these data indicate that vir genes do not play a major role in the strict sense of antigenic variation in which variant proteins are clonally expressed.
3. The function of vir genes What is the function of vir genes? Perhaps the answer to this fundamental question resides in the unique biology of P. vivax. Unlike P. falciparum, P, vivax does not sequester in the deep capillaries of inner organs and therefore infected reticulocytes circulate constantly through the spleen. The spleen is a lymphoid organ exquisitely adapted to selectively clear abnormal red blood cells, particles from the blood and infectious agents including malaria (Bowdler, 2002). Seminal studies from the group of Leon Weiss and collaborators have elegantly documented the structure and function of the spleen under normal and pathological
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conditions including experimental infections with different rodent malaria parasites in mice (Weiss, 1983, 1990, 1991; Weiss et al., 1985, 1986, 1989; Tablin et al., 2002). Thus, in non-pathological situations, the circulation of the spleen is predominantly open as evidenced by the lack of continuity of most arterial ends with venous structures and the presence in between them of a reticular meshwork. Moreover, the filtration capacity of the spleen is low as more than 90% of the blood flows through the spleen at similar rates as in any other tissues with conventional vasculature (Weiss, 1983). Upon malaria infection however, the spleen rapidly enlarges (splenomegaly) and there are striking changes in spleen cell distribution, including, among others, size increases of white pulp, development of germinal centres and replenish of the reticular meshwork with macrophages, plasma cells and erythroblasts (Weiss et al., 1986; Villeval et al., 1990; Yadava et al., 1996; Achtman et al., 2003). Moreover, in non-lethal murine models of malaria, the ‘open’ circulation of the spleen is suddenly and temporarily changed to a ‘closed’ circulation due to the formation of syncitial layers of contractile fibroblasts that form physical barriers, termed barrier cells (Weiss, 1990, 1991). 3.1. Barrier cells Barrier cells are contractile fibroblasts present in low numbers in normal spleens but whose number increases significantly in both human and mouse spleens in pathological conditions such as thalassemia, congenital spherocytic anemia, sickle sick disease, spectrin deficiency and malaria (Tablin et al., 2002). Of relevance for this review, is that two different phases have been characterised in non-lethal reticulocyte-prone malaria infections such as Plasmodium berghei strain 17X in BALB/c mice: a phase of a rapid increase in parasitemia (precrisis) followed by a rapid decrease in parasitemia (crisis). In these experimental infections barrier cells are able to by-pass the open circulation of the spleen by anastomosing the capillaries with the venules generating a ‘closed circulation’ (Weiss, 1990). Moreover, these cells physically surround reticulocytes allowing them to mature within the spleen before their release into circulation and protecting them from infected erythrocytes (Weiss, 1990). Although seemingly paradoxical, barrier cells render the enlarged spleen ‘aesplenic’ allowing infectious agents to freely circulate in blood and thus causing disease. Unexpectedly however, the numbers of infected red blood cells in non-lethal P. chabaudi infections augment instead of decrease during precrisis (Yadava et al., 1996). In striking contrast, formation of closed circulation is not detected in lethal murine malaria models such as Plasmodium yoelii strain 17XL in BALB/c mice (Weiss, 1990). 3.2. Spleen erythropoiesis Besides its major selective clearance function, the spleen is also an erythropoeitic organ which under stress conditions
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can be responsible for close to 40% of the RBC populations, and where reticulocytes are massively stored before release into circulation (Bowdler, 2002). Indeed, spleen erythropoiesis in humans has been documented in different pathological conditions, including malaria (Palitzsch et al., 1987; Beguin et al., 1989; Bowdler, 2002). Moreover, several studies using rodent models have demonstrated that upon non-lethal infection spleen-erythropoiesis replenishes the red pulp reticular meshwork with reticulocytes which, upon crisis, are released into circulation to compensate the anaemia associated with malaria. In contrast, there is an impairment of spleen erythropoiesis in lethal infections and this is suggested as a main cause of death (Villeval et al., 1990). 3.3. Plasmodium vivax malaria Plasmodium vivax malaria is a non-lethal disease characterised by relatively low parasitemia and pathogenicity presumably due to the fact that P. vivax invades preferentially, if not exclusively, reticulocytes (Kitchen, 1938; Galinski and Barnwell, 1995). In spite of this low parasitemia, however, recrudescence, reinfections and relapses of natural parasite populations result in continuous parasitemia. Moreover, anaemia is commonly associated with P. vivax malaria as reported from a very well controlled study on induced P. vivax infections in patients receiving treatment of neuro-syphilis between 1940 and 1963 (Collins et al., 2003). Thus, anaemia as judged by hematocrit values was detected in 85 out of 98 patients and hematocrit never recovered to normal values in these patients. Interestingly, it was calculated that on a 7-day infection, blood will be mostly depleted of uninfected reticulocytes (Collins et al., 2003).
4. Proposed function of vir genes in spleen-specific cytoadherence and clearance evasion On the basis of the data reviewed above, we propose the following hypothesis to explain the function of vir genes, how the spleen exerts its immunological functions and how P. vivax counters them. Plasmodium vivax is a reticulocyteprone, if not specific, non-lethal infection that is associated with anaemia in acute and chronic infections. Moreover, the existence of barrier cells and erythropoiesis in the human spleen has now been clearly demonstrated (Bowdler, 2002). Thus, under this scenario, upon infection with P. vivax and during precrisis, parasites induce anaemia, splenomegaly, spleen erythropoiesis with massive storage of reticulocytes and formation of barrier cells (Fig. 4). Barrier cells augment the filtration capacity of the spleen and protect spleen reticulocytes from invasion while maturing before their release into circulation during crisis. To avoid spleen clearance, P. vivax-infected reticulocytes specifically cytoadhere to barrier cells where they are protected from macrophage clearance and upon disruption of barrier cells during crisis, free merozoites will immediately encounter
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Fig. 4. Proposed hypothesis of the function of variant proteins in spleen-specific cytoadherence and clearance evasion. Plasmodium vivax infected reticulocytes specifically cytoadhere to barrier cells during precrisis protecting themselves from macrophage spleen-clearance. In parallel, spleen-erythropoiesis sustains maturation of large numbers of young reticulocytes in the red pulp before their release into circulation to compensate for the anaemia associated with vivax infections. During crisis (i.e. paroxisms which coincide with the rupture of schizont-infected reticulocytes), barrier cells no longer sustain a closed circulation allowing free merozoites to immediately invade new reticulocytes. Artery (A), periarterial lymphatic sheath (PALS), lymph node (LN), venous sinusoid (S). Adapted from Weiss (1990), Yadava et al. (1996), and Tablin et al. (2002).
uninfected reticulocytes in large numbers. It has not escaped our attention that infected-reticulocytes representing all the different asexual blood stages will always be found in circulation due to the fast flow circulation of the spleen (Bowdler, 2002). Thus, to avoid phagocytic destruction in other organs, parasites express non-clonally polymorphic proteins on the surface of infected reticulocytes to avoid formation of highly specific opsonizing antibodies. The discovery of highly polymorphic and conserved vir subfamilies in P. vivax suggest that they can have a dual role. Highly polymorphic vir subfamilies will protect infected reticulocytes from macrophage destruction whereas conserved vir subfamilies might act as specific ligands for barrier cells. Alternatively, the main and unique function of vir proteins is macrophage-clearance escape and parasite ligands specific for spleen barrier cells are yet to be identified. While our hypothesis remains speculative, it nevertheless makes clear predictions that can be assessed experimentally in non-human primates and rodent malaria models. Most relevant, we hope that this hypothesis together with a recent model on how P. vivax escape spleen-clearance (Suwanarusk et al., 2004), will further stimulate research on
the complex interactions of P. vivax parasites, reticulocytes and the spleen, which are central to a better understanding of how P. vivax escape the hosts’ immune response and establish a chronic infection.
Acknowledgements We are particularly grateful to Cassiano Pereira Nunes for figures and the drawing representing our hypothesis. CFB is a research associate supported by CAPES. SRM is a PhD student supported by CAPES. The laboratory of HAP receives support from FAPESP (01/09401-0) and CNPq (394651/90-7).
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Invited review
Variant genes and the spleen in Plasmodium vivax malaria Hernando A. del Portilloa,*, Michael Lanzerb, Sergio Rodriguez-Malagaa, Fidel Zavalac, Carmen Fernandez-Becerraa a
Departamento de Parasitologia, Instituto de Cieˆncias Biomedicas, Universidade de Sa˜o Paulo, Av. Lineu Prestes 1374, Sa˜o Paulo, SP 05508-900, Brazil b Hygiene-Institut, Abteilung Parasitologie, Universita¨t Heidelberg, Heidelberg, Germany c Johns Hopkins Malaria Research Institute, Johns Hopkins University, Baltimore, MD, USA Received 25 May 2004; received in revised form 18 October 2004; accepted 18 October 2004
Abstract It is generally accepted that Plasmodium vivax, the most widely distributed human malaria, does not cytoadhere in the deep capillaries of inner organs and thus this malaria parasite must have evolved splenic evasion mechanism in addition to sequestration. The spleen is a uniquely adapted lymphoid organ whose central function is the selective clearance of cell and other particles from the blood, and microbes including malaria. Splenomegaly is a hallmark of malaria and no other disease seems to exacerbate this organ as this disease does. Besides this major selective clearance function however, the spleen is also an erythropoietic organ which, under stress conditions, can be responsible for close to 40% of the RBC populations. Data obtained in experimental infections of human patients with P. vivax showed that anaemia is associated with acute and chronic infections and it has been postulated that the continued parasitemia might have been sufficient to infect and destroy most circulating reticulocytes. We review here the basis of our current knowledge of variant genes in P. vivax and the structure and function of the spleen during malaria. Based on this data, we propose that P. vivax specifically adhere to barrier cells in the human spleen allowing the parasite to escape spleen-clearance while favouring the release of merozoites in an environment where reticulocytes, the predominant, if not exclusive, host cell of P. vivax, are stored before their release into circulation to compensate for the anaemia associated with vivax malaria. q 2004 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: vir; Plasmodium vivax variant genes; Spleen; Reticulocytes; Cytoadherence; Barrier cells
1. Antigenic variation Antigenic variation refers to the capacity of many viruses, bacteria and parasites to alter their surface proteins in order to evade the immune system and establish chronic infections (Brown and Brown, 1965, Dietsch et al., 1997, Kyes et al., 2001). Malarial parasites establish chronic infections that persist for long periods of time, despite the concurrent presence of a strong immune response directed against parasite-encoded antigens exposed on the surface of the host erythrocyte (Miller et al., 1994; Kyes et al., 2001). The host’s efforts to eliminate the pathogen are counteracted by the parasite’s capability of constantly changing the * Corresponding author. Tel.: C55 11 3091 7209; fax: C55 11 3091 7417. E-mail address: [email protected] (H.A. del Portillo).
antigenic specificity of these proteins (Brown and Brown, 1965; Brown and Hills, 1974; Reeder and Brown, 1996). This cycle of parasite killing by variant specific antibodies and outgrowth of novel variants results in recurrent waves of parasitemias. As a direct result of antigenic variation, immunity to malaria develops only slowly and rarely progresses to a condition of complete protection even in highly endemic areas where re-infections leading to patent parasitemias occur frequently (Miller et al., 1994). Brown and Brown (1965) presented the first conclusive evidence in support of antigenic variation in malarial parasites. Their work relied on an observation made by Eaton in 1939 who found that erythrocytes infected with mature stages of Plasmodium knowlesi were agglutinated by sera from rhesus monkeys immune to P. knowlesi, but not by sera from normal rhesus monkeys (Eaton, 1938). Using the ability to agglutinate schizont-infected erythrocytes as
0020-7519/$30.00 q 2004 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2004.10.012
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a serological test (SICA test), Brown and Brown showed that, if a rhesus monkey was infected with a single strain of P. knowlesi and drug cured, serum collected 2 weeks later caused specific agglutination of erythrocytes infected with this parasite (Brown and Brown, 1965). When the monkey was reinfected with the same strain, however, the parasite population that was recovered was not recognised by the serum from the first convalescent blood. Experiments with clonal populations and their respective agglutinating antibodies further demonstrated the existence of antigenic variation in P. knowlesi and paved the way to the identification and characterisation of the variant antigens on the surface of infected erythrocytes (Howard et al., 1983). Antigenic variation is a regular feature of all Plasmodium species where it has been sought, including the simian malarias, P. knowlesi (Brown and Brown, 1965; Barnwell et al., 1982) and Plasmodium fragile (Handunnetti et al., 1987), the rodent malaria, Plasmodium chabaudi (McLean et al., 1982), and the human malarias, Plasmodium vivax (Mendis et al., 1988; del Portillo et al., 2001) and Plasmodium falciparum (Hommel et al., 1983; Biggs et al., 1991; Roberts et al., 1992). That all Plasmodium species apparently use antigenic variation clearly emphasises the importance of this immune evasion strategy in parasite survival, yet fails to explain why malarial parasites insert components into their host cell membranes in the first place, subsequently becoming a target for the host’s immune response. After all, the exposure of antigens on the erythrocyte looks like a suicidal action (Borst et al., 1995). It has been proposed that such deliberate parasiteprompted killing mechanism prevents fulminate parasite growth and subsequent host death, enhancing the parasite’s chances of being transmitted to another host (Saul, 1999). Increasing evidence point towards a different function of these variant antigens. When malarial parasites propagate within the erythrocyte, they alter their host cell (Aikawa, 1988). The spleen recognises abnormal erythrocytes, removes them from circulation and the erythrocyte, together with its intracellular parasite, are phagocytosed and destroyed by cells of the spleen’s reticuloendothelial system (Wyler, 1983). To avoid spleen-specific clearance the parasite needs to take action. A drastic measure is displayed by P. falciparum. This parasite, in particular its late developmental stages that cause the most damage to the host erythrocyte, avoid passage through the spleen by cytoadhesion to the endothelium of venular capillaries in the deep vascular bed of inner organs (Bignami and Bastianelli, 1889; Miller, 1969). Sequestration is mediated by the P. falciparum erythrocyte membrane protein 1 (PfEMP1), a variant antigen that renders the infected erythrocyte adhesive, allowing it to bind to a broad range of receptors on the surface of endothelial cells and uninfected erythrocytes (Baruch et al., 1995; Smith et al., 1995; Su et al., 1995). While sequestration appears to be the prime spleen evasion mechanism developed by P. falciparum, P. vivax, must have
found a different solution to avoid spleen clearance as it is widely accepted that it does not sequester in the deep vascular bed of inner organs having an obligate passage through this organ.
2. Antigenic variation in P. vivax Plasmodium vivax is the most widely distributed human malarial parasite and is estimated to cause 80–90 million cases a year and in Latin America and Asia, is the most prevalent species among the four human malarial parasites (Mendis et al., 2001). Plasmodium vivax cannot be continuously cultured in vitro, so parasites must be obtained from human patients or infected monkeys. Although this limitation has somewhat hindered research of this parasite species, it has also allowed researchers to obtain valuable information from natural parasite populations. Plasmodium vivax invades preferentially, if not exclusively, reticulocytes (Kitchen, 1938; Galinski and Barnwell, 1995). Interestingly, infected reticulocytes exhibit an ultrastructure distinct from that of P. falciparum infected erythrocytes. Whereas P. falciparum infected erythrocytes display electron-dense protrusions on their surface, called knobs, P. vivax infected reticulocytes suffer invaginations (Aikawa, 1988) and have different rheological properties that confer infected reticulocytes more deformability (Suwanarusk et al., 2004). Ultrastructural studies have identified these invaginations as caveola vesicle complexes. These structures are consistent with the Schuffner dots, small brick-red dots scattered over the host reticulocyte cytoplasm, observed in Giemsa stained thin blood smears (Schu¨ffner, 1899). The parasite induced invaginations of the host reticulocyte surface contain P. vivax-encoded antigens as demonstrated by immuno-electronmicroscopy and immunofluorescence using sera from P. vivax-infected patients (Matsamuto et al., 1988; Udagama et al., 1988). Significantly, these antigens are highly polymorphic. Thus, of 13 independent P. vivax isolates tested only some reacted with heterologous sera obtained from other P. vivax patients (Mendis et al., 1988). These results have suggested that P. vivax, like P. knowlesi, P. chabaudi and P. falciparum, use antigenic variation to establish a chronic infection. 2.1. Variant genes in P. vivax To identify gene(s) encoding variant antigens in P. vivax, we have constructed a representative genomic library of P. vivax in yeast as artificial chromosomes (YAC) and screened it for the presence of telomeric YACs (Camargo et al., 1997). Telomeric YAC clones were investigated because, in P. falciparum, subtelomeric domains contain clusters of multigene families, such as var, rif, stevor, and clag, which are implicated in antigenic variation and cytoadherence (Gardner et al., 1998; Bowman et al.,
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Fig. 1. Gene structure of virulent genes in Plasmodium falciparum, P. vivax and P. knowlesi. The two-exon (P. falciparum), three-exon (P. vivax), and 10-exon (P. knowlesi) (al-Khedery et al., 1999) structures of the var, rif, stevor, vir, and sicavar genes, are shown. Exons are depicted as boxes with introns as linking lines. Canonical signal sequences of rif and stevor genes are found in shaded boxes and predicted transmembrane domains as black boxes. Scale bars are in kilo-base pairs (kb).
1999). Several different clones were identified and one of them was completely sequenced (http://www.sanger.ac.uk/ Projects/P_vivax/). The sequence revealed the existence of a P. vivax-specific subtelomeric multigene family, termed vir (P. vivax variant genes), with ca. 600–1000 copies per haploid genome (del Portillo et al., 2001). Moreover, analysis of P. vivax chromosomes, as well as eight random telomeric YAC clones, revealed that members of this gene family are predominantly located within the subtelomeric domain of most, if not all, P. vivax chromosomes. These vir genes have a consensus three-exon structure which contrasts with the 10-exon structure of the sicavar genes of P. knowlesi and with the two-exon structure of var, rif and stevor genes of P. falciparum (Fig. 1). The 5 0 short exon lacks a predicted signal peptide sequence. The second exon is highly variant containing a predicted
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transmembrane domain and conserved cysteine residues. BLAST analysis of vir sequences from this exon predicted the existence of different subfamilies, termed A–F, varying in their extent of allele polymorphism. The third exon is of uniform size and encodes a cytosolic domain. The region covering the splice boundary between exons 2 and 3 is extremely well conserved. Similar to the P. knowlesi sicavar protein and the P. falciparum PfEMP-1 protein, typical signal sequences are lacking in vir antigens. A striking feature of the P. vivax subtelomeric domain is the lack of complex repeats. Apart from the telomeric repeat, there are no other large tandem arrays of repetitive sequence. This is in marked contrast to P. falciparum (Gardner et al., 2002) (Fig. 2). In fact a predicted gene occurs less than 1 kb from the telomeric repeat (del Portillo et al., 2001). Further hybridisation studies with a P. falciparum probe specific for subtelomeric repeats failed to cross-hybridise with nine random, non-contiguous P. vivax telomeric YAC clones, confirming that P. vivax chromosome ends lack complex repeat sequence elements (del Portillo et al., 2001). It has been postulated that complex repeat sequences facilitate ectopic recombination events between subtelomeric var genes in P. falciparum, by promoting the formation of a close, bouquet-like arrangement of ends from different chromosomes in the nucleus (Freitas-Junior et al., 2000). It is tempting to speculate that the high copy number of vir genes at chromosome ends itself promotes the pairing of, and recombination between, heterologous chromosomes, without the need of complex repeat sequence elements. Interestingly, in both P. vivax and P. falciparum, the genes involved in antigenic variation cluster within subtelomeric domains and their primary sequences are highly species specific. In contrast, internal chromosome regions appear highly conserved between P. falciparum and P. vivax (Tchavtchitch et al., 2001), consistent with the concept of a structural and functional compartmentalisation of Plasmodium chromosomes (Lanzer et al., 1993). It appears that
Fig. 2. Telomere organisation of virulent genes in Plasmodium vivax and P. falciparum. Scheme of chromosome-ends of P. falciparum and P. vivax displaying the telomere-associated repetitive DNA sequences and subtelomere-associated multigene families. Order and orientation of genes are shown. Exons are coloured boxes with introns as linking lines. (For interpretation of references to colour in this figure legend, the reader is referred to the web version of the article.)
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those genes encoding antigens located at the surface of the host cell have moved to the subtelomeric domain where they are subjected to high recombination frequencies. This in turn allows for the expansion of these genes into gene families and, at the same time, creates variants with novel antigenic phenotypes. Indeed, true multigene family orthologs of vir genes have recently been described in the subtelomeric regions of rodent and monkey malarias (Janssen et al., 2002). 2.2. Vir genes and antigenic variation Several lines of evidence initially suggested that vir genes were involved in antigenic variation in P. vivax: Firstly, analysis of expressed vir genes in natural parasite populations obtained from three different patients demonstrated that they were abundantly expressed. Furthermore, there are many different sets of variants expressed among different patients suggesting that P. vivax uses its vast repertoire of vir genes in natural parasite populations. Second, a preliminary serological survey with two different expressed vir gene tags showed that vir proteins were immunogenic and immunovariant. Third, laser confocal microscopy using fluorescently labelled affinity purified human anti-vir antibodies or anti-peptide antibodies specific for a conserved motif of vir proteins demonstrated their location at the surface of the infected reticulocyte (del Portillo et al., 2001). Together, these results established vir genes as the first virulent factor to be putatively involved in antigenic variation and chronic infections in P. vivax. To further study these vir genes, we are analysing the gene repertoire and expression of vir genes from individual patients in the Brazilian Amazon. To do so, parasite DNA and RNA are extracted from individual patients and used as templates for PCR and reverse transcriptase PCR amplifications of genomic and expressed vir sequences, respectively, using degenerate, vir-specific oligonucleotides representing subfamilies A–F. For instance, from one patient, we have generated a total of 45 different sequences (24 genomic and 21 transcribed) representing about 27 kb of vir sequences. In silico analysis of alignments made with CLUSTALX and manually edited with GeneDoc confirmed that vir genes are structured into different subfamilies varying in their extent of allele polymorphism in natural parasite populations (Fig. 3). Thus, there are highly polymorphic subfamilies such as A and C and highly conserved subfamilies such as D. Moreover, there was remarkably little overlap among all sequences clearly indicating the existence of a vast vir gene repertoire in natural infections. To determine the role of vir proteins in naturally acquired immunity we are analysing the naturally acquired humoral immune responses of P. vivax infected patients in the Brazilian Amazon using several different GST-vir tags. Results have confirmed that vir proteins are immunovariant; thus, immune sera from 23 out of 32 patients (w72%) specifically reacted against one or more of 22 GST-vir tags representing all vir subfamilies. Unexpectedly however, there was no significant difference in
Fig. 3. vir Genes are structured into conserved and highly polymorphic subfamilies in natural infections. Deduced amino acid alignments of genomic and expressed vir sequences obtained from parasites of an individual Plasmodium vivax patient were made with CLUSTALX and manually edited with GeneDoc (http://www.psc.edu/biomed/genedoc/). The unrooted tree was generated from these alignments using TreeView 32. Sequences were obtained from cloned PCR-fragments amplified with degenerate yet vir-specific subfamily oligonucleotides as described elsewhere (del Portillo et al., 2001).
the recognition of vir-tags by immune sera of first-infected or multiple-infected patients suggesting that several vir proteins are expressed on the surface of individual parasites. Laser confocal microscopy of individual infected reticulocytes using polyclonal monospecific antibodies against peptides representing different vir subfamilies have indeed proved so (Fernandez-Becerra C and del Portillo HA, unpublished). This data contrasts with similar studies in var genes of P. falciparum and with the sicavar genes of P. knowlesi (Kyes et al., 2001; Barnwell et al., 1982; Barnwell et al., 1983). Most importantly, together, these data indicate that vir genes do not play a major role in the strict sense of antigenic variation in which variant proteins are clonally expressed.
3. The function of vir genes What is the function of vir genes? Perhaps the answer to this fundamental question resides in the unique biology of P. vivax. Unlike P. falciparum, P, vivax does not sequester in the deep capillaries of inner organs and therefore infected reticulocytes circulate constantly through the spleen. The spleen is a lymphoid organ exquisitely adapted to selectively clear abnormal red blood cells, particles from the blood and infectious agents including malaria (Bowdler, 2002). Seminal studies from the group of Leon Weiss and collaborators have elegantly documented the structure and function of the spleen under normal and pathological
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conditions including experimental infections with different rodent malaria parasites in mice (Weiss, 1983, 1990, 1991; Weiss et al., 1985, 1986, 1989; Tablin et al., 2002). Thus, in non-pathological situations, the circulation of the spleen is predominantly open as evidenced by the lack of continuity of most arterial ends with venous structures and the presence in between them of a reticular meshwork. Moreover, the filtration capacity of the spleen is low as more than 90% of the blood flows through the spleen at similar rates as in any other tissues with conventional vasculature (Weiss, 1983). Upon malaria infection however, the spleen rapidly enlarges (splenomegaly) and there are striking changes in spleen cell distribution, including, among others, size increases of white pulp, development of germinal centres and replenish of the reticular meshwork with macrophages, plasma cells and erythroblasts (Weiss et al., 1986; Villeval et al., 1990; Yadava et al., 1996; Achtman et al., 2003). Moreover, in non-lethal murine models of malaria, the ‘open’ circulation of the spleen is suddenly and temporarily changed to a ‘closed’ circulation due to the formation of syncitial layers of contractile fibroblasts that form physical barriers, termed barrier cells (Weiss, 1990, 1991). 3.1. Barrier cells Barrier cells are contractile fibroblasts present in low numbers in normal spleens but whose number increases significantly in both human and mouse spleens in pathological conditions such as thalassemia, congenital spherocytic anemia, sickle sick disease, spectrin deficiency and malaria (Tablin et al., 2002). Of relevance for this review, is that two different phases have been characterised in non-lethal reticulocyte-prone malaria infections such as Plasmodium berghei strain 17X in BALB/c mice: a phase of a rapid increase in parasitemia (precrisis) followed by a rapid decrease in parasitemia (crisis). In these experimental infections barrier cells are able to by-pass the open circulation of the spleen by anastomosing the capillaries with the venules generating a ‘closed circulation’ (Weiss, 1990). Moreover, these cells physically surround reticulocytes allowing them to mature within the spleen before their release into circulation and protecting them from infected erythrocytes (Weiss, 1990). Although seemingly paradoxical, barrier cells render the enlarged spleen ‘aesplenic’ allowing infectious agents to freely circulate in blood and thus causing disease. Unexpectedly however, the numbers of infected red blood cells in non-lethal P. chabaudi infections augment instead of decrease during precrisis (Yadava et al., 1996). In striking contrast, formation of closed circulation is not detected in lethal murine malaria models such as Plasmodium yoelii strain 17XL in BALB/c mice (Weiss, 1990). 3.2. Spleen erythropoiesis Besides its major selective clearance function, the spleen is also an erythropoeitic organ which under stress conditions
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can be responsible for close to 40% of the RBC populations, and where reticulocytes are massively stored before release into circulation (Bowdler, 2002). Indeed, spleen erythropoiesis in humans has been documented in different pathological conditions, including malaria (Palitzsch et al., 1987; Beguin et al., 1989; Bowdler, 2002). Moreover, several studies using rodent models have demonstrated that upon non-lethal infection spleen-erythropoiesis replenishes the red pulp reticular meshwork with reticulocytes which, upon crisis, are released into circulation to compensate the anaemia associated with malaria. In contrast, there is an impairment of spleen erythropoiesis in lethal infections and this is suggested as a main cause of death (Villeval et al., 1990). 3.3. Plasmodium vivax malaria Plasmodium vivax malaria is a non-lethal disease characterised by relatively low parasitemia and pathogenicity presumably due to the fact that P. vivax invades preferentially, if not exclusively, reticulocytes (Kitchen, 1938; Galinski and Barnwell, 1995). In spite of this low parasitemia, however, recrudescence, reinfections and relapses of natural parasite populations result in continuous parasitemia. Moreover, anaemia is commonly associated with P. vivax malaria as reported from a very well controlled study on induced P. vivax infections in patients receiving treatment of neuro-syphilis between 1940 and 1963 (Collins et al., 2003). Thus, anaemia as judged by hematocrit values was detected in 85 out of 98 patients and hematocrit never recovered to normal values in these patients. Interestingly, it was calculated that on a 7-day infection, blood will be mostly depleted of uninfected reticulocytes (Collins et al., 2003).
4. Proposed function of vir genes in spleen-specific cytoadherence and clearance evasion On the basis of the data reviewed above, we propose the following hypothesis to explain the function of vir genes, how the spleen exerts its immunological functions and how P. vivax counters them. Plasmodium vivax is a reticulocyteprone, if not specific, non-lethal infection that is associated with anaemia in acute and chronic infections. Moreover, the existence of barrier cells and erythropoiesis in the human spleen has now been clearly demonstrated (Bowdler, 2002). Thus, under this scenario, upon infection with P. vivax and during precrisis, parasites induce anaemia, splenomegaly, spleen erythropoiesis with massive storage of reticulocytes and formation of barrier cells (Fig. 4). Barrier cells augment the filtration capacity of the spleen and protect spleen reticulocytes from invasion while maturing before their release into circulation during crisis. To avoid spleen clearance, P. vivax-infected reticulocytes specifically cytoadhere to barrier cells where they are protected from macrophage clearance and upon disruption of barrier cells during crisis, free merozoites will immediately encounter
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Fig. 4. Proposed hypothesis of the function of variant proteins in spleen-specific cytoadherence and clearance evasion. Plasmodium vivax infected reticulocytes specifically cytoadhere to barrier cells during precrisis protecting themselves from macrophage spleen-clearance. In parallel, spleen-erythropoiesis sustains maturation of large numbers of young reticulocytes in the red pulp before their release into circulation to compensate for the anaemia associated with vivax infections. During crisis (i.e. paroxisms which coincide with the rupture of schizont-infected reticulocytes), barrier cells no longer sustain a closed circulation allowing free merozoites to immediately invade new reticulocytes. Artery (A), periarterial lymphatic sheath (PALS), lymph node (LN), venous sinusoid (S). Adapted from Weiss (1990), Yadava et al. (1996), and Tablin et al. (2002).
uninfected reticulocytes in large numbers. It has not escaped our attention that infected-reticulocytes representing all the different asexual blood stages will always be found in circulation due to the fast flow circulation of the spleen (Bowdler, 2002). Thus, to avoid phagocytic destruction in other organs, parasites express non-clonally polymorphic proteins on the surface of infected reticulocytes to avoid formation of highly specific opsonizing antibodies. The discovery of highly polymorphic and conserved vir subfamilies in P. vivax suggest that they can have a dual role. Highly polymorphic vir subfamilies will protect infected reticulocytes from macrophage destruction whereas conserved vir subfamilies might act as specific ligands for barrier cells. Alternatively, the main and unique function of vir proteins is macrophage-clearance escape and parasite ligands specific for spleen barrier cells are yet to be identified. While our hypothesis remains speculative, it nevertheless makes clear predictions that can be assessed experimentally in non-human primates and rodent malaria models. Most relevant, we hope that this hypothesis together with a recent model on how P. vivax escape spleen-clearance (Suwanarusk et al., 2004), will further stimulate research on
the complex interactions of P. vivax parasites, reticulocytes and the spleen, which are central to a better understanding of how P. vivax escape the hosts’ immune response and establish a chronic infection.
Acknowledgements We are particularly grateful to Cassiano Pereira Nunes for figures and the drawing representing our hypothesis. CFB is a research associate supported by CAPES. SRM is a PhD student supported by CAPES. The laboratory of HAP receives support from FAPESP (01/09401-0) and CNPq (394651/90-7).
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