Antigenic Variation & Immune Evasion

ANTIGENIC VARIATION & IMMUNE EVASION

Fondation Mérieux Conference Center “Les Pensières” Veyrier-du-Lac - France

March 30 - April 1, 2009

Steering Committee:

• Simona BARZU

• Alister CRAIG

• Catherine DUTEL

• Bernard IVANOFF

• Christophe LONGUET

• José Juan LOPEZ-RUBIO

• Jacques LOUIS

This meeting was made possible through unrestricted educational grants from Sanofi Pasteur.

Welcome Letter

March 30, 2009

Dear Participant,

It is our pleasure to welcome you to the symposium entitled:

“Antigenic Variation and Immune Evasion”

in Fondation Mérieux’s Conference Center “Les Pensières.” We hope you will enjoy this meeting, which brings together some of the world’s foremost experts.

The format of the discussion is intended to generate discussion and interaction among participants and to foster the dissemination of new information on this topic. The conference will provide an opportunity for specialists to exchange their knowledge and experience through collaboration with researchers from around the world.

Over the next three days, the team at Les Pensières will be on hand to help you with any questions you may have and to make your stay and conference as comfortable and valuable as possible.

Benoît Miribel Directeur Général Fondation Mérieux

For more information: www.fondation-merieux.org

Background and rationale

Multicellular organisms possess very sophisticated defense mechanisms that are designed to counter the continual microbial insult of the environment within the vertebrate host. The interaction between a pathogen and the mammalian immune system resulted in two basic transmission patterns. In the first one, pathogens replicate rapidly and are transmitted early, before the development of an effective immune response. The second transmission pattern is typified by persistent infection until there is an opportunity for transmission, which might not occur until long after a fully mature and effective immune response has developed. Evasion of host immune defenses may be achieved by antigenic variation, which is common in vector-borne and mucosally-transmitted pathogens. Although strain-to-strain variation in antigenic molecules (antigen diversity) is common, antigenic variation refers to a single strain specifically changing a subset of its antigens either to sustain ongoing infection or re-infect hosts even though the first infection was successfully cleared. Antigenic variation concerns proteins or glycoproteins (i) expressed at the surface of extracellular patho - gens or at the surface of cells infected by intracellular ones, (ii) involved in adhesion or invasion to host cells or in cytoadhesion of infected cells, and (iii) often immunodominant. In parasites and bacteria, antigenic variation sensu stricto often involves specific multigene families devo- ted to that function and generally occurs at rates orders of magnitude faster than gradual accumulation of genetic alterations associated with random, non targeted mutational events. The genetic information for producing a family of antigenic variants is available in the cell but only one variant is expressed at a given time. Switching between members of a multigene family involves a complicated mechanism of activation and silencing. The mechanisms underlying antigenic variation are either based on transcriptional and epigenetic control (in situ switching), like in Plasmodium falciparum or Giardia lamblia, or on gene conversion (unidirectional recombination), described in parasites (Trypanosoma brucei, Babesia bovis) and bacteria (Borrelia hermsii, Neisseria gonorrhoeae, and Anaplasma marginale). A major conceptual issue is how sequential dominance of antigenic variants is achieved by a population of parasites within a host, each independently expressing a variant. Several theories have been developed, mainly from studies of Trypanosoma, Plasmodium and Borrelia. Exceptions to these rules have been recently demonstrated: organisms with diverse antigenic variants may co-exist within the same host (Treponema pallidum, M. genitalium) and the mechanism for antigenic variation may be reciprocal recombination, as opposed to gene conversion (M. genitalium). Antigenic variation also occurs in viruses, facilitating, in addition to immune escape or immune subversion mechanisms, persistent infections. Variation is not based on switching between members of multigene fami lies, but rather on spontaneous mutations during viral replication. Examples are lentiviruses (HIV, FIV), HCV (hepatitis C virus), caliciviruses. Infection with one isolate also leads to multiple antigenic variants within one host. For some of the pathogens cited, which represent dramatic disease burdens within human or animal populations, numerous vaccine approaches have been tested with limited success. The immunodominant character and the roles in pathogenesis of the variant antigens represent difficult challenges. In the face of the highly efficient antigenic variation strategy of the immunodominant unique surface protein VSG, vaccines for protection against Trypanosoma brucei are not contemplated. Some 20 candidates are currently in development at different phases for malaria prophylaxis. Moreover, virulence evolution in response to vacci nation needs to be considered during vaccine development. Genome sequencing of bacterial and parasite pathogens opens however new perspectives for prophylactic approaches. Immune evasion mechanisms other than antigenic variation (based on host immune system regulation or subversion) will not be discussed in this meeting (they were the main topic of previous meetings).

Scientific Program

Monday March 30, 2009

17h30 - 18h30 Registration

18h30 - 18h45 Welcome Address Christophe LONGUET

Keynote Lecture : Antigenic varia- 18h45 - 19h15 Etienne PAYS tion as adaptive process 19h45 Dinner

Tuesday March 31, 2009

Session 1 Parasites and bacteria: antigenic variation through transcriptional control Chaired by Lars Hviid and Chris Newbold

Epigenetic regulation of antigenic 08h30 - 08h50 Jose Juan LOPEZ RUBIO variation in Plasmodium falciparum 08h50 - 09h10 Discussion

Constrains of antigenic variation 09h10 - 09h30 on the development of vaccines Alister CRAIG against Malaria 09h30 - 09h50 Discussion

09h50 - 10h30 Coffee break

Giardia lamblia mechanisms and 10h30 - 10h50 Theodore NASH vaccines 10h50 - 11h10 Discussion

Ehrlichia canis, E.chaffeensis, 11h10 - 11h30 their p28 paralogs, and mechanism David WALKER of persistence

Scientific Program

11h30 - 11h50 Discussion

12h00 - 14h00 Lunch

Session 2 Parasites and bacteria: antigenic variation through gene conversion Chaired by David Barry and David Soll

African trypanosomes: can variant surface glycoprotein gene conver- 14h00 - 14h20 Richard MC CULLOCH sion be considered a target for disease intervention? 14h20 - 14h40 Discussion

Gene conversion in Babesia bovis 14h40 - 15h00 antigenic variation and its implica- David ALLRED tions for vaccine development 15h00 - 15h20 Discussion

15h20 - 15h50 Coffee Break

Gene conversion in bacteria: the 15h50 - 16h10 Alan BARBOUR Borrelia models 16h10 - 16h30 Discussion

Implication of antigenic variation 16h30 - 16h50 for vaccine development: the Ana- Kelly BRAYTON plasma marginale model 16h50 - 17h10 Discussion

19h00 Dinner

Scientific Program

Wednesday April 1, 2009

Session 3 Antigenic variation in viruses Chaired by Luisa Figueirido and James Mullins

Consequences of antigenic varia- 08h30 - 08h50 Bluma BRENNER tion in terms of treatment in HIV 08h50 - 09h10 Discussion

09h10 - 09h30 Feline Caliciviruses Hervé POULET

09h30 - 09h50 Discussion

09h50 - 10h20 Coffee Break

Humoral response against 10h20 - 10h40 Dimitri LAVILLETTE Hepatitis C virus 10h40 - 11h00 Discussion

Molecular basis on antigenic 11h00 - 11h20 Bette KORBER variation in HIV 11h20 - 11h40 Discussion

12h00 - 14h00 Lunch

Consequences of antigenic 14h00 - 14h20 Brigitte AUTRAN variation in terms of vaccines 14h20 - 14h40 Discussion

Scientific Program

Session 4 Microbial evolution in response to vaccination Chaired by Luc Vanhamme

Evolution of pathogens in 14h40 - 15h00 Vitaly GANUSOV response to vaccination 15h00 - 15h20 Discussion

15h20 - 15h40 The case of Malaria Margaret MACKINNON

15h40 - 16h00 Discussion

16h00 - 16h20 Conclusion

16h20 End of the meeting

Keynote Lecture

Antigenic variation as adaptive process ...... Antigenic variation as adaptive process

...... Etienne PAYS Université Libre Bruxelles - Belgium ......

...... Antigenic variation is classically presented as a way for parasitic organisms to escape the immune defences of their hosts. In the ...... case of African trypanosomes, this process is particularly important, as the entire surface of the cell is regularly changed after only a ...... few days. Moreover, the variant antigenic surface is strikingly ho- mogeneous, as it consists in a single glycoprotein species (VSG, for Variant Surface Glycoprotein) present in 10 million copies. With ...... such an obvious antigen presentation, it is clear that the system is not meant to avoid the immune reaction, but rather to trigger a res- ...... ponse for the benefit of the parasite. This benefit is the continuous control of the parasite burden, which allows infection to persist for ...... long periods in a given host until cyclical transmission by tsetse flies permits the escape of some parasites and their eventual propaga- ...... tion to new hosts. The aim of the talk is to illustrate how VSG switching is only a facet of a more comprehensive system that confers ...... adaptation of trypanosomes to a large variety of mammalian hosts. Two examples will be detailed: the VSG-linked variation of the VSG-like transferrin receptors and the VSG-linked expression of ...... the VSG-like protein allowing Trypanosoma brucei rhodesiense to resist lysis by human serum......

......

Session 1

Parasites and bacteria: antigenic variation through transcriptional control ...... Epigenetic silencing of virulence gene families in malaria parasites ...... Jose Juan LOPEZ-RUBIO ...... Institut Pasteur - France

...... Malaria parasites have developed sophisticated strategies that al- ...... low phenotypic variation without affecting the genotype. This way, malaria parasites can cope with changing host environment such ...... as adaptive immune response and increase the duration of infection in the human host. This is generally achieved by the expression of clonally variant molecules either at the erythrocyte membrane ...... or merozoite surface. Switching of expression to another variant molecule avoids immune clearance and prolongs the period of in- ...... fection. In Plasmodium falciparum, the causal protozoan agent of the most severe form of human malaria, antigenic variation is me- ...... diated by sequential expression of single variant surface molecules termed PfEMP1 encoded by approximately 60 var genes. In- ...... creasing evidences illustrate that the complex process of antigenic variation is orchestrated by epigenetic factors. Recently, we have ...... shown that different histone modifications are enriched at the 5´ flanking and coding regions of active, poised or silenced var genes. A genome wide analysis of the trimethylation at lysine 9 of histone ...... H3, a histone mark linked to silenced var genes, reveals a restricted association of this mark with var genes and other virulence gene ...... families. Therefore, our ChIP-on-chip results demonstrate that a specific repressive histone mark is constrained to variant surface ...... antigen families in P. falciparum, which locate mainly at subtelome- ric ends and at a few chromosomal internal positions. Interestingly, ...... we show that all H3K9me3 enriched loci locate to the nuclear peri- phery, implying chromosome loop formation. Our work establishes ...... a link between the nuclear architecture and the restriction of histone marks to specific genome regions. Furthermore, disruption of the ...... histone deacetylase PfSir2 causes changes in H3K9me3 that are discontinuous along chromosomes and associated with disrupted monoallelic transcription. Our data point to the existence of peri- ...... nuclear repressive centers associated with control of expression of malaria parasite genes involved in phenotypic variation and patho- ...... genesis.

......

...... Constraints of antigenic variation on the development of vaccines against malaria ...... Alister CRAIG ...... Liverpool School of Tropical Medicine - UK

...... The human malaria parasite Plasmodium falciparum has develo- ...... ped a complex mechanism of antigenic variation that allows it to alter the major surface proteins on the infected erythrocyte. This ...... is seen in natural infections as an acquisition over several years of serum responsiveness to the infected erythrocyte surface leading ...... relatively rapidly to protection against severe disease but rarely to sterile protection. The main candidate for a variant surface antigen (VSA)-based vaccine has been the protein PfEMP1, which is en- ...... coded by the var gene family. Although each parasite genome has only 50-60 var genes the population repertoire is much larger, and ...... so the concept of an anti-PfEMP1 vaccine is difficult to entertain. However various groups have identified restricted variation asso- ...... ciated with pathology, particularly in pregnancy-associated malaria but also in paediatric disease. Molecular studies have also started ...... to define structurally conserved regions of PfEMP1 involved in the adhesive interactions mediated by this VSA. Thus, despite the high ...... degree of variation seen in this protein and the practical difficulties associated with immuno-epidemiological studies there are indica- tions that a VSA-based vaccine to control some aspects of disease ...... (rather than infection per se) might be possible......

......

...... Antigenic Variation in Giardia llamblia

...... Theodore NASH NIH - USA ......

...... Giardia lamblia consist of at least 7 related groups and of these twocommonly infect humans, Assemblage A (Groups 1 and 2) and ...... Assemblage B( AssB) (Group 3). AssA and AssB have significant biological and biochemical differences, dissimilar variant-specific ...... surface protein (VSPs) repertories, and significantly distinct genomes and sequence variability that therefore should be considered different species. All Giardia lamblia and likely G. muris undergo ...... surface antigenic variation of VSPs. These consist of a family of cysteine rich-proteins proteins that have similar structure and mo- ...... tifs most notably a zinc finger motif, a GGCY containing motif(s) and a conserved carboxyl transmembrane domain terminating in ...... an absolutely conserved CRGKA cytoplasmic tail. The tail is post translationally modified and palmitoylated at the cysteine and ci- ...... trullinated at the arginine. During vegetative growth in culture, each trophozoite expresses only one VSP on its surface and switching ...... occurs spontaneously at varying rates depending upon the Ass and VSP ranging from once every 6.5-13 generations. There is no ...... preferred sequence but a slight preference for some VSPs. VSP switching occurs in an Assemblage A prototype but not in the AssB during encystation/excystation. In vitro VSPs are processed and re- ...... leased into the medium. The mechanisms involved in antigenic variation are incompletely known but are epigenetic in nature. Recent ...... studies showed that RNAi posttranscriptional mechanisms control VSP expression whereby all vsps are transcribed but only one vsp ...... transcript is expressed. The natural course of Giardia infections in humans is not well documented. Giardia infections are commonly ...... prolonged and at least up to day 21 experimental infections suggest that antigenic variation plays a role in parasite survival. In mice ...... both humoral and biological selection pressures determine which VSPs can expressed. Humoral responses to VSPs develop early and are immunodominant. They are inhibitory and/or cytotoxic and ...... most likely eliminate parasites expressing encountered VSPs in the host. IgA antibodies in animals play an essential role in preventing ...... and controlling infection. In addition biological selection also occurs. Giardia expressing particular VSPs can be expressed in SCID ...... mice and highly immunosuppressed gerbils and the favored VSPs differ among hosts...... Suite

...... Despite ability to control infection, protective immunity in humans appears to be either limited or easily circumvented, because almost ...... all treated persons in a highly endemic region are rapidly reinfec- ted indicating poor immunity or immunity that can be easily circu- ...... mvented, perhaps through antigenic variation. How important are VSPs in development of protective immunity? Although this cannot ...... directly be answered, animal data infers that immunity to VSPs and as well as other antigens can be partially protective. Although the ...... development of immunity to all Giardia VSPs present in a particular community would seemingly confer protection, this may not be the case. VSP repertoires differ, and therefore not protective for a ...... dissimilar VSP group, VSPs possibly mutate, and immunity may be partial or varying, wane or not be able to respond to very low ...... numbers of organisms. Dr. Lujan proposes to produce parasites for vaccination by knockdown of Dicer, a procedure that allows pan ...... expression of VSPs. Although there is no doubt about the pathogenicity of Giardia in most developed countries where epidemics and ...... sporadic disease are well documented, there is no consensus that Giardia causes measurable disease in highly endemic regions......

......

...... Ehrlichia canis, E. chaffeensis, E. muris, Their p28 Para- logs, and Mechanisms of Persistence ...... David H. WALKER ...... University of Texas - USA

...... The genus Ehrlichia includes the agents of prevalent, emerging hu- ...... man infectious zoonoses (E. chaffeensis, life threatening human monocytotropic ehrlichiosis; E. ewingii, a granulocytotropic ehrli- ...... chiosis affecting immunocompromised persons) and of classic ve- terinary diseases (E. canis, canine monocytic ehrlichiosis; E. rumi- ...... nantium, heartwater). Ehrlichia are small tick-transmitted obligately intracellular bacteria with a small circular genomes (1.2 -1.5 Mbp) and reside in an early endosome of monocytes or neutrophils. They ...... have a developmental cycle in which the smaller dense core cell is infectious but does not replicate and the larger reticulate cell repli- ...... cates but is not infectious. Ehrlichiae avoid immune clearance and cause persistent infections in their natural vertebrate hosts and are ...... acquired and maintained transstadially but not transovarially by the vector tick...... Ehrlichiae are gram negative bacteria that lack lipopolysaccharide ...... and peptidoglycan, but acquire cholesterol from the host that is in- corporated into their cell membranes for structural stability. Ehr- lichial proteins associated with host-pathogen interactions inclu- ...... ding twelve tandem repeat proteins and seven proteins that have eukaryote-like ankyrin repeats have been identified. In addition, ...... Ehrlichia possess a locus that encodes 16-25 major outer membrane proteins of 25-30 kDa that contain three major hypervariable ...... regions that are predicted to be surface exposed. Their diversity at the amino acid level ranges from 19 to 80% for E. chaffeensis...... It was widely speculated that sequential expression of these genes would mediate immune evasion. However, most of them are ...... actively transcribed simultaneously in cell culture, and dogs infected with E. chaffeensis produce antibodies to unique peptides re- ...... presenting each of the p28s relatively early in the infection. These observations indicate that the p28s are produced, stimulate the immune system, and do not result in immune evasion. A possible ...... species-specific function of these proteins is suggested in the predominant expression of p28-19 and p28-20 in mammalian cells and ...... of p28-14 in tick cells.

...... Suite ...... Ehrlichiae evade immune clearance by stealthily avoiding acti- ...... vation and downregulation of TLR2, TLR4, CD14, and IL-12 and decreasing innate immunity mediated by IL-15, IL-18, chemokine ...... receptors, and IL-8 receptor. Ehrlichiae are able to block the antimi- crobial effect of IFN-γ by inhibition of the Jak/Stat signaling pathway ...... leading to ehrlichial iron starvation. Further host defense evasion phox includes active blocking of O2- generation by degradation of p22 ...... triggered by E. chaffeensis binding to cell surface proteins and inhi- biting endosomal-lysosomal fusion by downregulation of transcription of vesicle docking proteins, Rab5, syntaxin 16, and SNAP23, ...... via a two component system (histidine kinase and a response re- gulator). Ehrlichiae have also evolved mechanisms of manipulating ...... the host cell to the advantage of the bacteria including inhibition of apoptosis to prolong the opportunity for intracellular growth...... The means by which a colony of bacteria that reside within a cyto- ...... plasmic vacuole can control specific host cell processes appear to include an ehrlichial type IV secretion system that is hypothesized ...... to transfer ehrlichial effector proteins that interact with host cell signaling, transcriptional regulation and vesicle trafficking and other ...... host mechanisms. Modulation of these processes has been linked to interactions between a secreted ehrlichial effector protein, p47, with host cell proteins polycomb group ring finger 5 protein, Src ...... protein tyrosine kinase FYN, protein tyrosine phosphatase non-receptor type 2, and adenylate cyclase-associated protein 1. Further- ...... more, an ankyrin repeat containing protein, p200 is translocated to the host cell nucleus where it interacts most likely via a host protein ...... with Alu-containing promoters, binding sites for transcription factors, and 5’URRs that mediate translation inhibition. The targets of ...... p200 include the genes for TNF-α, which plays a pathogenic role in the toxic shock-like illness, and CD48, a receptor for bacterial entry ...... involving caveolae, both of which are strongly upregulated during E. chaffeensis infection of monocytes. Thus the data indicate that ehrlichiae have evolved many mecha- ...... nisms to disarm the host defenses permitting these bacteria to sur- vive in their natural vertebrate host as long as the host lives rather ...... than by sequentially changing their antigens to avoid immune clearance......

......

Session 2

Parasites and bacteria: antigenic variation through gene conversion ...... African trypanosomes: can Variant Surface Glycoprotein gene conversion be considered a target for disease ...... intervention?

...... Richard MCCULLOCH University of Glasgow - UK ......

...... The basis for antigenic variation and immune evasion in the Afri- can trypanosome is a dense surface coat, composed of around 5 x 6 ...... 10 Variant Surface Glycoprotein (VSG) dimers, which covers the surface of this flagellated protistan parasite, shrouding invariant surface antigens from immune recognition and preventing com- ...... plement-mediated lysis. Antigenic variation involves switches in the identity of the expressed VSG, a process that is spontaneous, ...... can occur at rates around 103-fold higher than background mutation and displays a hierarchy, with distinct VSG types being observed ...... to be activated predominantly at the early, middle or late stages of chronic trypanosome infections. Such VSG switching draws upon ...... a huge archive of VSG genes. The size of this archive was revea- led by the genome sequence of one strain of Trypanosoma brucei, ...... TREU927, in which 940 VSGs have been annotated from a total estimated to be around 2000. The VSGs are found in chromosome subtelomeres, predominantly in arrays in megabase-sized chromo- ...... somes. Remarkably, the huge majority (86%) of these array genes are pseudogenes, and only a small fraction (4.5%) are functionally ...... intact. At any one time, only a single VSG is expressed, from a telomeric, multigene transcription unit termed the VSG expression site ...... (ES). Perhaps surprisingly, multiple ESs are found in the genome, and directed cloning and sequencing has shown that the numbers ...... vary between T. brucei strains and subspecies (from 23 to perhaps as low as 5). The multiplicity of ESs allows some VSG switching to ...... occur by transcriptional mechanisms, where the active ES is transcriptionally silenced and a silent ES is activated in a co-ordinated ...... reaction. Nevertheless, the predominant route for VSG switching is recombination reactions that move genes from the silent archive into the ESs...... Activation of a silent VSG by recombination in African trypanoso- ...... mes can occur by cross-over (reciprocal) recombination, but most VSG activations occur by gene conversion, generating a copy of ...... the silent VSG and moving it to the ES, where it replaces the resi- dent VSG. It is not clear that this is a single mechanism, however...... The earliest VSGs to be activated during an infection are directly telomeric, in particular those found in specialised mini-chromosomes. This does occur by gene conversion, but could also allow the use of break-induced replication, though this has not been tested genetically. In the hierarchy of activation, intact array VSGs are next activated, and the gene conversion of these is referred to as dupli- cative transposition...... Suite

...... Finally, late in infections, multiple VSG pseudogenes are combined to generate novel VSG mosaics by segmental gene conversion ...... reactions. Analysis of the factors that underlie VSG switching suggest that it is dependent on homologous recombination, the univer- ...... sally conserved pathway for repair of DNA double strand breaks and replication completion. Mutation of three key factors, RAD51, ...... BRCA2 and RAD51-3 (a RAD51 paralogue), causes significant reduction in the efficiency of VSG switching. However, it is unlikely ...... that a single recombination reaction acts, since any of the mutants cause a reduction, not abolishment, in VSG switching. This suggests that other, genetically uncharacterised, recombination reac- ...... tions can contribute, which could reflect the multiplicity of VSG switching mechanisms. For instance, the available assays for switching ...... analyse only the early events, so we cannot yet say if segmental gene conversion is also RAD51-dependent...... The above picture describes our understanding of the complexity of ...... antigenic variation in T. brucei, including the timing and hierarchy of VSG activation during an infection, the potential consequences of ...... widespread segmental gene conversion and the apparent use of a conserved DNA repair pathway to catalyse VSG switching. On the- ...... se bases, we will discuss the potential for using VSG gene conversion to provide a target for disease intervention, since it seems in- creasingly unlikely that the VSG coat can be considered a viable ...... vaccine candidate, at least against mammalian-infective parasites......

......

...... Gene conversion in Babesia bovis antigenic variation and its implications for vaccine development ...... David R. ALLRED ...... University of Florida - USA

...... Bovine babesiosis due to Babesia bovis is an often lethal acute ...... disease that progresses to an asymptomatic but highly persistent infection, marked by effective immunity to further disease. To ...... achieve persistence, this intraerythrocytic parasite cytoadheres to the capillary and post-capillary venous endothelium, presumably to avoid splenic passage. This capability requires the parasite-derived ...... protein, variant erythrocyte surface antigen-1 (VESA1). Antibody recognition of VESA1 abrogates in vitro cytoadhesion and may re- ...... sult in in vivo elimination of parasitized erythrocytes recognized by antibodies. The adhesive phenotype is protected by rapid antigenic ...... variation of both VESA1 subunits (1a and 1b), encoded by ves1α and ves1β genes of the ves multigene family. Previously, segmen- ...... tal gene conversion of the transcriptionally active ves1α and ves1β genes was demonstrated to be a major mechanism of VESA1 anti- ...... genic variation, resulting in the formation of mosaic subunits. Thus, extreme variability occurs in both subunits of this heterodimeric protein, with the total potential variation approximated by the product ...... of individual subunit variation. In addition to ves genes, the smorf gene family also possesses attributes highly suggestive of antige- ...... nic variation, although this has not yet been formally demonstrated. The extreme antigenic variability of VESA1 suggests it is an unlikely ...... candidate for vaccine development. However, at least three strategies targeting this protein could provide the host immune system ...... the opportunity for significant parasite control. Firstly, a strategy of immunizing with molecular mimics of the VESA1 binding surface ...... conceptually could induce an immune response capable of disrupting the adhesive function of a significant proportion of VESA1 iso- ...... forms. Secondly, a strategy of disrupting the mechanisms responsible for modification or switching of ves genes could reduce the ability of the parasite to hide from an ongoing immune response...... Thirdly, a strategy of disrupting the trafficking to and assembly of this protein in the erythrocyte membrane, could prevent the adhe- ...... sive function and render antigenic variation of VESA1 moot. Supported by NIAID grant #R01 AI055864......

...... Antigenic Variation in Relapsing Fever and Lyme Borre- liosis ...... Alan BARBOUR ...... University of California - USA

...... Relapsing fever (RF) and Lyme borreliosis (LB) are two tick-borne ...... diseases caused by members of the spirochete genus Borrelia. The two sets of agents have made different adaptations to their envi- ...... ronments and circumstances. RF spp. are transmitted by ticks that feed for only a short time, and this is associated with high densi- ...... ties of the spirochetes in the blood during infection. The window for transmission from a vertebrate host to a tick for a RF spp., such as B. hermsii, is prolonged by multiphasic antigenic variation, in ...... which a full-length gene for a surface protein is replaced at a single expression site by another allele in the genome. In its enzootic ...... locations the population structure of B. hermsii is limited in strain diversity. Vertical transmission of RF spp. is common...... In contrast, LB spp., such as B. burgdorferi, are transmitted by ticks ...... that feed for days at a time. In a vertebrate reservoir, such as small rodents, B. burgdorferi is primarily an infection of the skin and not ...... the blood. Antigenic variation occurs in the tissue phase of the infection, and only part of the variable gene is replaced by a cas- sette. In locations of LB transmission the population structure of B...... burgdorferi is quite diverse, with evidence of frequency-dependent selection for strain-specific antigens. The reservoir hosts undergo ...... serial infections with different strains. Vertical transmission of LB spp. does not occur...... Vaccines against LB have been developed. The most successful ...... to date are directed against a protein, OspA, that is usually expressed by B. burgdorferi only when it is in a tick. This protein does ...... not encounter the vertebrate immune system and, perhaps as a consequence, there are few polymorphisms across a wide variety ...... of strains. Antibodies of the vaccinee kill the spirochetes in the mid- gut of the blood-feeding tick before they have a chance to migrate into the host. Immunity against RF natural conditions is largely di- ...... rected against the variable antigens. A non-variable, tick-associated protein that is analogous to OspA has not to date been identified ...... in RF spp.

...... Implication of antigenic variation for vaccine development: the Anaplasma marginale model ......

...... Kelly BRAYTON Washington State University - USA ......

...... Anaplasma marginale is a rickettsial pathogen of ruminants that serves as an excellent model to study antigenic variation. A. mar- ...... ginale has a minimalist genome of 1.2 Mb and makes efficient use of it’s genome to produce a combinatorial array of surface protein ...... variants through gene conversion. The antigenic variation system is relatively simple, comprised of two variable molecules, MSP2 and MSP3, each encoded by a small gene family consisting of a ...... single expression site and a set of 5-10 donor alleles, depending on the strain. Each full length expressed molecule is composed of ...... a central hypervariable region (HVR) flanked by conserved termini, and the donor alleles contain the central HVR and truncated flan- ...... king regions. The small set of donor alleles allows for mapping of the expression site variants back to their donor alleles. We have ...... shown that expression site variants early in infection typically reflect the intact donor alleles, while complex mosaic variants, made from ...... multiple donor alleles, are selected for immune evasion during persistent infection. These mapping experiments illustrate the impor- tance of the donor allele repertoire: when an intact donor allele is ...... used in the expression site it confers a growth advantage to the or- ganism, and strains that contain novel or distinct donor allele(s) are ...... capable of establishing infection in an already infected host in the face of robust immunity to the initial infecting strain. These studies ...... indicate that the donor allele repertoire is under selection pressure to maintain function yet to be as diverse as possible to provide an ...... epidemiological advantage. Analysis of the allelic repertoire from seven strains reveals that many alleles recur in multiple strains, ...... suggesting that a cocktail of HVR alleles may provide a vaccination strategy that could protect against initial infection when the patho- ...... gen surface reflects the donor allele variants.

......

Session 3

Antigenic variation in viruses ...... Population Growth, Urban Concentration, Vulnerable Po- pulations and Regions Worldwide ...... Bluma BRENNER ...... Mc Gill University - Canada

...... The failure to introduce antiretroviral therapy (ART) into the resour- ...... ce-poor nations of Africa and Asia has led to the uncontrolled spread of HIV/AIDS and an unprecedented diversification of the pande- ...... mic to include 11 subtypes, 34 circulating recombinant forms and innumerable recombinant mosaics. There is an increased global ...... awareness of the need to tackle the global HIV-1 pandemic and to facilitate the introduction of ART into resource-poor settings. While cumulative findings from clinical studies suggest that antiretroviral ...... therapy will be of benefit in the overall treatment of non-B subtype infections, the 10-15% sequence diversity in reverse transcriptase ...... and protease, may contribute to inter-subtype differences in responses to the three major drug classes, including nucleoside and ...... non-nucleoside reverse transcriptase inhibitors, as well as protease inhibitors. Combination treatment regimens must be optimized to ...... minimize costs, preclude toxicities and limit emergent drug resistance in different subtypes...... Subtype C infections, presently account for 52% of global pandemic. Our laboratory has focussed on the analysis of subtype C sequence variations that may influence treatment response and emergent ...... drug resistance. Cell culture experiments show the selective rapid emergence of resistance in subtype C only. This resistance could ...... be ascribed to a subtype C signature valine codon polymorphism (GTA→GTG, subtype B vs. C), absent in other non-C viral subty- ...... pes. This polymorphism facilitates the single nucleotide transition to V106M (GTG→ATG) upon selective drug pressure with EFV but ...... not NVP. The V106M mutation has been observed in ~30% of patients harbouring subtype C infections failing efavirenz regimens...... Selection studies also show that a KKK codon motif in subtype C at codons 64, 65 and 66 favours the emergence of the K65R re- ...... sistance with many nucleoside analogues, including d4T, ddI, AZT, and tenofovir. Clinical trials in Africa sadly confirm the significant emergence of the K65R pathway in subtype C leading to treatment ...... failure with nucleoside analogues, particularly with suboptimal d4T and ddI regimens. In Mali, the rapid selection of the K65R and thy- ...... midine analogue mutations precludes effective second-line alterna- tive treatment regimens for a large patient population. Enzymatic ...... studies show that selection of K65R is driven by the nucleotide sequence of the subtype C reverse transcriptase that favours pau- sing. This new template-driven form of drug resistance may expand our understanding of HIV enzymatic processes...... Suite ...... Cumulative findings suggest subtype differences in responses to ...... single dose nevirapine, used to reduce vertical transmission to newborns. The overall risk of infant transmission at birth and 6-8 ...... wks following birth was 50% lower for subtypes A and D versus C. The rapid advent of resistance in mothers does not detract from the ...... benefit of NVP prophylaxis, especially when used in combination with ZDV and 3TC...... In the coming years, increasing numbers of persons having non-B infections exist will have the benefit of various combination treat- ments. It may be possible to optimize management of major non-B ...... subtype infections, particularly subtypes A, CRF01_AE, CRF02_ AG, and C in resource-poor settings. The promising hope for the ...... future study is illustrated in recent Rwanda, Zambia and Uganda studies that conclude that ART in conjunction with counselling may ...... markedly reduce rates of HIV transmission.

......

...... Antigenic variation in Feline Caliciviruses

...... Hervé POULET Mérial - France ......

...... Feline caliciviruses (FCV) are important pathogens of cats. They usually cause an acute, oral and respiratory disease. The preva- ...... lence of FCV infection in the cat population is high (~20%) despite vaccination. Vaccines can reduce clinical signs but do not prevent ...... infection and viral excretion. Recently, several outbreaks of virulent systemic disease (VSD) with a high mortality rate (~50%) have been reported in the USA and in Europe...... Caliciviruses are small positive-sense RNA non-enveloped viruses ...... with an icosaedral capsid. The capsid is made of 90 dimers of the major protein, VP1. VP1 has two domains, S and P. The P2 sub- ...... domain, the most external part of the P domain, protruding from the capsid surface, is involved in receptor binding and is the target of ...... neutralising antibodies. It contains two hypervariable regions (HVR) corresponding to the immunodominant antigenic sites. Structural ...... analyses suggested that the N-terminal HVR is strongly involved in the binding to JAM-1...... In vitro and in vivo studies have shown the emergence of neutralization-resistant mutants. In the field, the ability of the classical ...... vaccine strains to induce cross-neutralizing antibodies against circulating viruses has progressively decreased. The highly virulent ...... viruses responsible for the recent outbreaks of systemic disease belong to the population of FCV variants that are not neutralized ...... by vaccine-induced antibodies. The amino acid sequences of VSD- FCV and classical FCV strains were compared by using a multiple ...... correspondence analysis (MCA) that combined the main properties of the amino acids at specific sites. The MCA analysis discrimina- ...... ted most of the VSD-FCV and classical FCV strains, even if some strains could not be clearly differentiated. The most discriminative amino acid positions were found mainly but not only in the N-termi- ...... nal HVR. Some of the positions were previously shown to accept an amino acid substitution allowing escape from neutralization by ...... antibodies (in vitro assays with monoclonal antibody selection on classical FCV strains). Those changes were shown to affect the ...... usage of the JAM-1 receptor.

...... One of the challenges for FCV vaccines is the high antigenic variability of the circulating strains. Most vaccine strategies to overcome antigenic variability have been based on identification of strains with a broad antigenic relatedness, and/or combination of antigens from different strains. Lessons learnt from FCV and FCV vaccines may be relevant to human noroviruses...... Humoral response against Hepatitis C virus

...... Dimitri LAVILLETTE ENS Lyon - France ......

...... Hepatitis C virus (HCV) infection is a major health problem, affecting 2-3% of the population worldwide. 40% of chronic infected per- ...... sons develop end-stage liver cirrhosis and 60% develop hepato- cellular carcinoma. There is so far no vaccine against HCV and the ...... current antiviral therapy is expensive, relatively toxic and effective in only half of treated patients. Spontaneous viral clearance occurs in about 10%-20% of acutely infected individuals and results in re- ...... solution of infection without sequelae. However, the mechanisms leading to viral clearance or persistence are not well understood...... One difficulty is that acute phase HCV infection is often asymptomatic. As a result, many investigations have been confined to the ...... study of chronically infected patients. Acute infection has been stu- died primarily in chimpanzees, the only animal model susceptible ...... to HCV infection, and in recent years on small cohorts of patients (drug addicts, persons exposed to contaminated blood) to study ...... the anti-HCV immune response in the first weeks of infection. The immunological events during this early phase are crucial. The qua- ...... litative and quantitative characterization of the antiviral immune response in the blood as in the liver compartment, early in infection in the resolved HCV infections, and its comparison to that observed ...... during chronic infections could enable the identification of immunological events leading to the elimination or viral persistence. As for ...... other viruses (HBV; EBV, LCMV), both viral and host factors appear to play an important role for resolution of acute infection. Weak cel- ...... lular immune T cell, CD4 and CD8 (in terms of proliferation, cyto- toxicity of production of IFN-γ and IL-2), and a Th2 type responses ...... orientation cytokine profile are usually reported to be a factor of viral persistence. However, for a long time, due to the lack of convenient ...... neutralization assays, the role of humoral immune response has been difficult to address. Experiments and observations conducted to the notion that neutralizing antibodies existed, however their role ...... in the viral clearance or viral escape were elusive. In recent years, the development of robust tissue culture model ...... systems for HCV entry and infection has finally allowed study of antibody-mediated neutralization and to gain further insights into ...... viral targets of host neutralizing responses. Taken together, these studies suggest that viral escape from antibody-mediated neutrali- ...... zation occurs on several levels, including the high variability of the HCV genome and limited induction of cross-neutralizing antibodies; the association of HCV with serum factors such as LDL and very low-density lipoprotein (VLDL); the interplay of HCV glycoproteins and SR-BI with HDL and direct cell-to-cell transfer of the virus...... Suite

...... Even if clearance of HCV may be mediated by an orchestrated interplay of cellular and neutralizing immune responses, the eluci- ...... dation of escape mechanisms from adaptive immune responses are crucial for the understanding of HCV pathogenesis as well as ...... the development of novel preventive and therapeutic strategies to control HCV infection......

......

...... Molecular basis on antigenic variation in HIV

...... Bette KORBER Los Alamos National Laboratory - USA ......

...... Abstract not provided

......

...... Consequences of antigenic variation in terms of vaccines ...... Brigitte AUTRAN ...... Hôpital Pitié Salpêtrière - France

...... Abstract not provided ......

......

Session 4

Microbial evolution in response to vaccination ...... Evolution of pathogens in response to vaccination

...... Vitaly V. GANUSOV Los Alamos National Laboratory - USA ......

...... Vaccination is one of the greatest medical achievements of the last century. Many pathogens including smallpox and polio has been ...... brought under control in the world due to development of highly effective vaccines. However, for many pathogens, mainly those cau- ...... sing persistent infections such as HIV, malaria and tuberculosis, we have failed to develop effective vaccines. Those vaccines that are currently being developed to control HIV and malaria will most likely ...... not prevent infection of vaccinated hosts although may reduce the incidence of the disease. It is generally believed that the main effect ...... of the use of imperfect vaccines is potential adaptation of pathogens to avoid recognition by the vaccine-induced immune response. Whi- ...... le this can certainly be the case, inefficient, imperfect vaccines may also affect evolution of pathogens such as to increase or decrease ...... the level of exploitation of their hosts, or virulence. Using a novel theoretical approach, we investigate how vaccines that reduce the ...... rate of pathogen replication within the host (anti-growth vaccines) or vaccines that reduce the rate of pathogen transmission (anti-trans- ...... mission vaccines) from the infected host may affect the evolution of pathogen virulence. For pathogens causing only acute infections and as such being eventually cleared from the host, we find that ...... the use of either anti-growth or anti-transmission vaccines leads to the evolution of pathogens with an increased within-host growth ...... rate. Importantly, infection of unvaccinated hosts with such evolved pathogens results in high host mortality and as the consequence, in ...... low pathogen transmission. Vaccination of only a fraction of hosts with anti-growth vaccines may prevent pathogens from evolving ...... high virulence due to pathogen adaptation to unvaccinated hosts and thus protection of vaccinated hosts from pathogen-induced di- ...... sease. In contrast, anti-transmission vaccines may be beneficial only if they are effective enough to cause pathogen extinction. Our results suggest that particular mechanisms of action of vaccines ...... and their efficacy are crucial in predicting long-term evolutionary consequences of the use of low efficacy (imperfect) vaccines......

......

...... Vaccination and virulence evolution in malaria

...... Margaret MACKINNON KEMRI-Wellcome Trust Collaborative Programme - Kenya ......

...... One theory of why some pathogens are virulent (i.e. they damage their host) is that they need to extract resources from their host in ...... order to compete for transmission to new hosts, and this resource extraction can damage the host. Here we describe our studies in ...... malaria that test and support this idea. We have also showed that host immunity can exacerbate selection for virulence and therefore that vaccines that reduce pathogen replication may select for more ...... virulent pathogens, thereby eroding the benefits of vaccination and putting the unvaccinated at greater risk. While this theory is foun- ...... ded on continuous trait-based evolutionary principles, it can also apply to antigenic type-based evolution if the relationships between ...... antigenic type and intrinsic virulence are known. For most antigeni- cally variable pathogens, this is not the case. Thus empirical stu- ...... dies are required to determine whether the strains dominating the populations are the most virulent, and whether there are trade-offs ...... between pathogenicity and antigenic novelty, as appears to be the case for the clonally switching antigens in the most virulent of the ...... human malaria parasites, Plasmodium falciparum.

......

Speakers & Chairs

ALLRED David IVANOFF Bernard University of Florida Fondation Mérieux [email protected] [email protected]

AUTRAN Brigitte LAVILLETTE Dimitri Hôpital Pitié Salpêtrière ENS Lyon [email protected] [email protected]

BARBOUR Alan LONGUET Christophe University of California Fondation Mérieux [email protected] [email protected]

BARRY Dave LOPEZ-RUBIO Jose Juan University of Glasgow Institut Pasteur [email protected] [email protected]

BARZU Simona LOUIS Jacques Sanofi Pasteur Fondation Mérieux [email protected] [email protected]

BRAYTON Kelly MACKINNON Margaret Washington State University KEMRI [email protected] [email protected]

BRENNER Bluma MCCULLOCH Richard Mc Gill University University of Glasgow [email protected] [email protected]

CRAIG Alister MULLINS James Liverpool School of Tropical Medicine University of Washington [email protected] [email protected]

FIGUEIRIDO Luisa NASH Theodore Rockfeller University NIH [email protected] [email protected]

GANUSOV Vitaly PAYS Etienne Los Alamos National Laboratory Université Libre Bruxelles [email protected] [email protected]

HVIID Lars POULET Hervé Centre for Medical Parasitology Mérial [email protected] [email protected]

Speakers & Chairs

SOLL David University of Iowa [email protected]

VANHAMME Luc Université Libre Bruxelles [email protected]

WALKER David University of Texas [email protected]