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Rabies virus vaccines: Is there a need for a pan-lyssavirus vaccine?
Vaccine 30 (2012) 7447–7454
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Review
Rabies virus vaccines: Is there a need for a pan-lyssavirus vaccine? Jennifer S. Evans a,b , Daniel L. Horton a , Andrew J. Easton b , Anthony R. Fooks a,c , Ashley C. Banyard a,∗ a
Animal Health and Veterinary Laboratories Agency, Woodham Lane, Weybridge, Surrey, KT15 3NB, United Kingdom University of Warwick, Gibbet Hill Road, Coventry, West Midlands, CV4 7AL, United Kingdom c National Consortium for Zoonosis Research, University of Liverpool, Leahurst, Neston, South Wirral, CH64 7TE, United Kingdom b
a r t i c l e
i n f o
Article history: Received 14 August 2012 Received in revised form 5 October 2012 Accepted 7 October 2012 Available online 19 October 2012 Keywords: Lyssaviruses Glycoprotein Rabies Vaccines Protection
a b s t r a c t All members of the lyssavirus genus are capable of causing disease that invariably results in death following the development of clinical symptoms. The recent detection of several novel lyssavirus species across the globe, in different animal species, has demonstrated that the lyssavirus genus contains a greater degree of genetic and antigenic variation than previously suspected. The divergence of species within the genus has led to a differentiation of lyssavirus isolates based on both antigenic and genetic data into two, and potentially a third phylogroup. Critically, from both a human and animal health perspective, current rabies vaccines appear able to protect against lyssaviruses classified within phylogroup I. However no protection is afforded against phylogroup II viruses or other more divergent viruses. Here we review current knowledge regarding the diversity and antigenicity of the lyssavirus glycoprotein. We review the degree of cross protection afforded by rabies vaccines, the genetic and antigenic divergence of the lyssaviruses and potential mechanisms for the development of novel lyssavirus vaccines for use in areas where divergent lyssaviruses are known to circulate, as well as for use by those at occupational risk from these pathogens. Crown Copyright © 2012 Published by Elsevier Ltd. All rights reserved.
Contents 1. 2. 3. 4. 5. 6. 7.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7447 Genetic divergence within the lyssavirus genus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7448 Diagnostic evaluation of lyssavirus infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7450 Genetic and antigenic relationships within the lyssavirus genus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7450 Human rabies vaccines and cross protection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7451 Novel experimental vaccine formulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7451 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7453 Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7453 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7453
1. Introduction Rabies virus (RABV) is a member of the lyssavirus genus, a group of zoonotic pathogens within the family Rhabdoviridae that are of global importance as the cause of a fatal encephalitic disease. RABV is the archetypal lyssavirus and historically is one of the most feared viruses known to man and the only virus that is invariably fatal following the development of clinical disease [1,2]. The genome organisation of members of the lyssavirus genus is relatively simple, encoding just five genes that are transcribed from the non-segmented negative strand RNA genome and translated to produce five viral proteins: the nucleocapsid protein
∗ Corresponding author. Tel.: +44 1932 357722. E-mail address: [email protected] (A.C. Banyard).
(N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and the polymerase protein (L) [3]. The lyssavirus G protein is responsible for attachment and entry into host cells and is considered to be a major determinant of pathogenicity [4–6]. Rabies virus is the most extensively characterised virus within the lyssavirus genus and circulates in volant and non-volant mammalian populations across both the developing and developed world. It is estimated that RABV causes between 24,000 and 93,000 human deaths annually [7] although this figure is considered to be an underestimate [8,9]. Critically, the majority of human deaths attributed to lyssavirus infection occur in the developing world where the virus continues to circulate within dog populations [7,10], with the highest disease burden being in the Indian subcontinent and Africa [11]. While both pre- and post exposure prophylaxis tools have been available to prevent disease caused by RABV for over 30 years these are often unavailable in areas
0264-410X/$ – see front matter. Crown Copyright © 2012 Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.vaccine.2012.10.015
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J.S. Evans et al. / Vaccine 30 (2012) 7447–7454
Fig. 1. Bayesian phylogenetic analysis of glycoprotein ectodomain sequences from representative lyssavirus species, implemented in BEAST (v1.4.8). The GTR+I model of nucleotide substitution and a chain length of 5 million were used. The maximum clade credibility tree was chosen using Treeanotator (v1.4.8) and viewed using Figtree (v1.1.2) estimated samples sizes were >100 and posterior probabilities at key nodes are shown. Scale bar represents substitutions per site. Accession numbers for each of the lyssavirus species analysed in this study are: ABLV AF801020, RABV CVS-11 EU352767, RABV Pitman Moore AJ871962, RABV Flury-HEP GU565704, BBLV JF311903, KHUV EF614261, EBLV-2 EF 157977, ARAV EF614259, DUVV EU293119, EBLV-1 EU293109 and EU293120, IRKV EF614260, MOKV HM623780 and EU293118, SHIBV GU170201, LBV ‘B’ EF547431, LBV ‘C’ EF547425, LBV ‘D’ GU170202, LBV ‘A’ EF547432, WCBV EF614258 and IKOV JN800509.
where the virus is endemic. There are a suspected 10 million potentially infectious contacts between humans and rabid animals each year which makes rabies an important target for prevention and control strategies [12]. Alongside infection attributed to RABV, other members of the lyssavirus genus are able to cause a fatal encephalitic disease in humans. The majority of lyssavirus species have been isolated from bat populations and whilst infection with RABV is documented, the true burden due to the other lyssaviruses remains largely undefined within both human and animal populations, notably due to a lack of diagnostic assays that are able to differentiate between lyssavirus species [13]. Here we review the current status of rabies vaccines, the protection they afford against different lyssavirus species and the potential burden of other lyssaviruses on animal and human health. Finally, we comment on the need for pan-lyssavirus vaccines to protect both those at risk through local endemnicity of divergent viruses in different populations as well as those at risk through occupational activities. 2. Genetic divergence within the lyssavirus genus The lyssavirus genus is divided into 12 species, the majority of which are predominantly associated with infection in bats (Fig. 1). Within Europe, alongside RABV in terrestrial wildlife populations three other lyssavirus species have been genetically characterised: European Bat Lyssavirus type-1 (EBLV-1), EBLV-2 and Bokeloh Bat Lyssavirus (BBLV). EBLV-1 is further genetically separated into two lineages which show an overlap in geographical incidence. EBLV-1a is found predominantly throughout northern Europe whilst EBLV1b has been detected in southern Germany, France and Spain. Both EBLV-1 lineages are associated with infection of Eptesicus fuscus, the Serotine bat although infection of other bat species such as the Meridional Serotine (E. isabellinus) has suggested the circulation of a third lineage of EBLV-1 [14,15]. EBLV-1 cross species transmission (CST) events have occurred at a very low frequency but with no maintenance of infection within the new host [16–18]. In contrast, EBLV-2 is predominantly associated with infection of
Myotis daubentonii, the Daubenton’s bat, across much of northern Europe and no cross species transmission events have been reported although two fatal human cases have occurred [19,20]. There has been only a single isolate of BBLV, obtained from a Natterer’s bat (Myotis natterei) in Germany in November 2009 [21]. In other parts of the world several other, predominantly bat associated lyssaviruses occur [15]. In Australia, Australian bat lyssavirus (ABLV) has been isolated from five different bat species since its initial isolation in 1996 [22] and has also been implicated in human fatalities [23,24]. A further four Eurasian lyssaviruses have been described, predominantly from single virus isolates from different bat species. These include Aravan virus (ARAV) isolated from a bat in Kyrgystan [25], Khujand virus (KHUV) isolated in Tajikistan [26], Irkut virus (IRKV) isolated from a bat in Eastern Siberia [25] and West Caucasian Bat virus (WCBV) isolated from a bat in the Caucasus mountains in Russia [25]. Phylogenetic classification of these Eurasian isolates have shown them to be distinct species within the lyssavirus genus with WCBV being the most genetically divergent [27]. Of these, only Irkut virus has been associated with human fatality [28]. Across Africa a number of bat lyssaviruses have been detected. The most frequently reported is Lagos bat virus (LBV) which has been isolated from multiple bat species distributed across subSaharan Africa [29]. There are currently four defined lineages of LBV; lineage A consists of Kenyan (2007), Senegalese (1985) and French (1999) isolates, lineage B contains only the original Nigerian isolate, lineage C contains isolates from Zimbabwe, South Africa and the Central African Republic and lineage D contains a single isolate identified in an Egyptian fruit bat in Kenya [30,31]. It has been suggested that Lagos bat virus isolates could be subdivided into several genotypes based on criteria adopted by the ICTV whereby all isolates belonging to the same species have identities of greater than 80%. LBV isolates are more divergent than this across each lineage (Fig. 1) and as such may ultimately be re-classified. Duvenhage virus (DUVV) was initially isolated from a fatal human infection following a bat bite and was subsequently isolated from a number of bat species, mainly Nycteris species in South Africa
Table 1 Nucleotide and amino acid identity of the lyssavirus G protein. Percentage identities are shown. Where identity is less than or equal to 60% they are shaded green. Viruses that have been tentatively classified into phylogroups are shaded accordingly.
Amino acid (% identity) ABLV
LBVD
LBVB
WCBV
48
52
58
56
58
48
50
47
47
53
52 81
LBVC
RABV RV61
DUVV
EBLV 1
73
75
71
73
72
47
48
46
48
48
50
50
50
50
51
77
57
57
57
58
74
55
56
57
59
79
73
57
58
56
80
74
56
58
57
72
59
59
58
58
58 90
SHIBV
MOKV
58
60
58
48
49
48
54
52
53
53
80
82
78
79
81
77
85
IKOV
52
WCBV
55
55
LBVA
59
53
56
LBVF
59
52
58
73
LBVB
59
53
57
73
73
LBVC
58
54
58
72
74
77
SHIBV
60
55
58
71
71
72
72
RABV CVS
MOKV
57
52
57
69
71
69
67
67
RABV CVS
70
53
56
58
59
58
59
60
58
RABV RV61
71
53
54
59
59
60
60
61
59
87
DUVV
67
52
58
58
60
58
58
59
59
65
65
EBLV 1
68
53
57
60
62
59
60
62
61
66
68
BBLV
EBLV 2
KHUV
76
76
76
77
46
47
48
47
51
52
51
51
59
56
57
56
57
58
57
57
58
57
57
59
58
56
57
57
58
59
58
57
57
57
59
58
58
59
57
59
57
58
58
57
57
57
57
69
70
68
73
75
73
75
71
73
69
75
77
76
78
80
75
78
78
79
77
80
84
78
80
79
78
75
76
76
83
85
83
87
86
73
IRKV
ARAV
IRKV
66
53
57
59
60
60
61
60
60
66
67
70
74
ARAV
70
53
57
58
61
60
60
61
60
70
69
70
74
70
BBLV
71
54
58
58
60
60
60
60
60
70
70
71
72
70
74
EBLV 2
71
53
57
58
61
61
61
60
60
70
71
72
74
70
76
77
KHUV
73
53
57
58
60
60
59
59
60
69
70
71
72
75
75
76
J.S. Evans et al. / Vaccine 30 (2012) 7447–7454
Nucleotide (% identity)
ABLV
LBVA
IKOV
88 79
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J.S. Evans et al. / Vaccine 30 (2012) 7447–7454
and Zimbabwe. There have been two further cases of DUVV infection in humans, both of African origin. Shimoni bat virus (SHIBV) was first isolated in 2009 from a Commerson’s leaf-nosed bat in Kenya [31]. Phylogenetically, SHIBV lies between Mokola virus and WCBV (Fig. 1). Mokola virus (MOKV) is an African lyssavirus which is only one of two lyssavirus species that has never been detected within bat species. MOKV has been isolated from a range of mammals including a shrew, domestic cats and a dog. However, sera from LBV positive bats has been shown to neutralise MOKV making assessment of the range of species infected with MOKV difficult and as a result bats cannot be ruled out as a reservoir for MOKV. There have been two recorded human cases of MOKV infection [32]. The latest lyssavirus to be identified is Ikoma lyssavirus (IKOV). IKOV was isolated from an African civet and is only the second non-rabies lyssavirus to have been isolated only from terrestrial carnivore species although its origin within chiropteran species is assumed from the circumstances within which it was detected [33]. IKOV is genetically divergent from all the other lyssaviruses [34]. 3. Diagnostic evaluation of lyssavirus infection Reporting of RABV infection remains problematic across areas of the developing world where the disease is endemic and as a result the estimated annual death toll is believed to be an underestimate of the true burden of disease. However, where virus infection is detected, the current diagnostic assays are unable to differentiate between RABV and the other lyssaviruses. Currently, the ‘Gold Standard’ OIE prescribed test for lyssavirus diagnosis is the fluorescent antibody test (FAT) [35]. This test is only performed on post mortem tissues as it requires a brain smear with polyclonal antibodies used to detect virus antigen. Unfortunately, whilst the antibodies used in the FAT are able to detect all lyssaviruses isolated, they are unable to differentiate between them. Indeed, the most recently identified lyssavirus, IKOV, was characterised following RT-PCR and sequence analysis of antigen positive brain material from terrestrial carnivores in Tanzania. Without the application of genetic characterisation this lyssavirus species would not have been discovered [33]. There have been very few reports describing isolates from human or animal fatalities that have been genetically typed as being caused by lyssaviruses other than RABV. Where this has occurred, initial diagnostic tools have merely confirmed infection with a lyssavirus. Importantly, for many of these infections, even if pre-immunisation of the individual had occurred, depending on the infecting virus, a protective antibody titre may not have been generated. 4. Genetic and antigenic relationships within the lyssavirus genus Across the lyssavirus genus, viruses are classified according to both genetic and antigenic data. Recently, there has been a shift in nomenclature from the definition of lyssavirus genotypes to that of lyssavirus species [36]. In addition to the separation of genetically distinct viruses into species, these viruses can also be divided into phylogroups using antigenic data [36,37]. This differentiation is mainly based on the degree of cross protection afforded by the current rabies vaccines between each of the viruses characterised. Genetic data allow reliable quantitative comparison of lyssavirus divergence [38] and sequence data are available for at least one isolate from all 14 virus species either already classified within the lyssavirus genus or awaiting classification. Across the open reading frame (ORF) encoding the G protein the lyssaviruses are at most 50% divergent at the nucleotide level while sharing only 46% identity at the amino acid level (Table 1). Genetically, IKOV is the most divergent lyssavirus [33]. However, using genetic data to predict
Fig. 2. Schematic representation of the lyssavirus G protein. Defined antigenic sites and conserved cysteine residues are shown. C residues are coloured according to proposed linkages within the mature protein [71]. TM: transmembrane; NH2: amino terminus; COOH: Carboxy-terminus.
vaccine cross protection is limited by the unpredictable effect of amino acid substitutions on antigenic variation, depending on their nature and location. Measuring antigenic variation directly typically relies on less reliable serological assays, which are affected by individual variation in antibody response. Consequently, serological data are considered to have low resolution, and are often only used qualitatively. The improved resolution of antigenic maps generated using antigenic cartography linked to serological assay data has allowed more precise comparison of antigenic and genetic data for pathogens including influenza viruses [38]. Application of these antigenic cartography techniques to a global panel of lyssaviruses has allowed integration of genetic and antigenic data [27]. On average a 4.8% change in the G protein ectodomain amino acid sequence caused a change of one antigenic unit between viruses (equivalent to a two-fold change in antibody titre). Although there is a good correlation (95% CI for r = 0.81–0.88) between genetic distance in the glycoprotein and antigenic distance, over 30% of antigenic variation could not be predicted by genetic distance alone. Investigation of discrepancies between genetic and antigenic distances showed, for example, that IRKV and ARAV are antigenically more closely related to EBLV-2 than EBLV-1, and that KHUV is closer to classical RABV than would be expected from
J.S. Evans et al. / Vaccine 30 (2012) 7447–7454
phylogenetic relationships. LBV and MOKV are antigenically distant and have been placed in a different phylogroup based on genetic distance and limited cross neutralisation [27,39] (Fig. 1). There is a low degree of cross neutralisation between phylogroups I and II and also between both phylogroups and WBCV [27]. In vivo vaccine challenge experiments have shown reduced or no efficacy of current licensed rabies vaccines against viruses in phylogroup II (MOKV, LBV, SHIBV) [40] and phylogroup III (WCBV) [41]. There is also evidence for variable vaccine efficacy against some viruses in phylogroup I [41,42] which suggests a gradual loss of vaccine protection corresponding to antigenic distance from vaccine strains. Improving prediction of vaccine protection using glycoprotein sequence alone would be a significant advance for future vaccine development and also for assessing the possible threat posed by novel lyssaviruses. However the improved accuracy requires more detailed knowledge of the individual antigenic effects of amino acid substitutions. Estimates of the individual antigenic effect of substitutions can be made for example, using known antigenic sites, protein structure and amino acid properties. It is now possible to test those estimates using site directed mutagenesis and antigenic cartography. The lyssavirus G protein which is present on the surface of the virion is the major viral component responsible for induction of a host antibody response and is the target of host neutralising antibodies [43]. The regions of the G protein responsible for the differences in antigenicity seen within what is otherwise a rather conserved genus are ill defined. The G protein is relatively conserved in length across the genus with an ORF encoding between 523 and 534 amino acids. Furthermore all lyssavirus proteins contain 14 highly conserved cysteine residues alongside the antigenic domains that contribute to the structure of the protein. Currently, defined antigenic domains within the G protein include four major antigenic sites and one minor antigenic site (Fig. 2). Historically, these have been defined through scanning mutagenesis experiments and mapping of monoclonal antibody (mAb) binding sites [44]. The positions and a consensus amino acid sequence for each antigenic site are detailed in Table 2. Antigenic site I is a region containing both conformational and linear epitopes as it is delineated by the monoclonal antibody mAb 509–6 that recognises a conformational epitope as well as antibody CR57 that recognises a linear epitope [45]. Antigenic site II has two domains, IIa and IIb so is a discontinuous conformational epitope. Antigenic site III is continuous, though no site specific mAbs are able to bind unfolded protein which indicates that this epitope forms part of a loop on the protein surface and it is this tertiary structure which enables recognition by antibodies or of neuronal receptors [45]. Antigenic site IV consists of only two amino acids and is continuous though it contains overlapping linear epitopes. Minor site ‘a’ is located in close proximity to site III but contains no overlapping epitopes and consists of only two amino acids [45].
5. Human rabies vaccines and cross protection From a public health perspective, the efficacy of rabies vaccines against infection with other lyssaviruses is of great interest to both the scientific and human health communities [1]. All rabies vaccines licensed for use within the developed world consist of inactivated preparations of live attenuated classical rabies virus strains. The provision of protection against the African and Eurasian lyssaviruses remains a problem. Human fatalities have highlighted occasions where death can be attributed to a non-RABV lyssavirus (Table 3). Vaccination and challenge studies in animal models suggest that rabies vaccines provide protection against
7451
DUVV [42], the EBLVs [46] and some of the recently identified Asian lyssaviruses [41]. However, LBV infection in rabies vaccinated companion animals has highlighted the lack of protection against non-rabies lyssaviruses following vaccination [47,48]. Furthermore, pre- and post-exposure vaccination failed to prevent disease and death in an animal model of WCBV infection [41]. These factors suggest that more cross reactive vaccine formulations may be necessary in areas where a threat to the human population comes from non-rabies lyssaviruses. Recent advances in the antigenic characterisation of different lyssaviruses may also aid future cross-reactive vaccine design [27]. The level of cross protection against IKOV is unknown although sequence comparisons suggest that there will be little or no cross protection afforded by current rabies vaccines against this novel lyssavirus [32]. Few human cases involving infection with non-rabies lyssaviruses have been reported although this may be due to the lack of discriminatory diagnostic tools without which the true burden remains unknown [49,50].
6. Novel experimental vaccine formulations The question of defining protection afforded against different lyssaviruses has been the basis for a number of studies into the development of recombinant viruses or glycoproteins as potential novel vaccine strategies. All approaches to date have focussed on the generation of vaccines that protect against phylogroup I and phylogroup II viruses [1,39]. Mebatsion et al. [51] used a reverse genetics system to generate recombinant viruses containing both heterologous and chimeric lyssavirus G proteins and, using reporter gene expression, showed that these could assemble and function as infectious virions. This study used a MOKV G protein as a heterologous replacement for the wildtype G protein in a RABV full length clone. The G gene of MOKV encodes a 523 amino acid protein, 2 amino acids shorter than that present in RABV with only 56.7% amino acid identity. No sequence homology was identified between the signal sequences or transmembrane and cytoplasmic domains of MOKV and RABV. However, the ectodomains of these two viruses (amino acids 19–430) had 62% amino acid identity although the five defined antigenic sites are poorly conserved (Table 2). Despite this, from a structural perspective all 14 cysteine residues were conserved between these viruses suggesting a similarity at the structural level. Expressing the MOKV G in the RABV full length clone allowed virus rescue and growth. Also, infectious virus was recovered when a chimeric G protein containing the ectodomain or a short surface epitope was substituted for the wildtype RABV G protein. This demonstrated that the cytoplasmic domain of MOKV was able to interact heterotypically with the wildtype RABV proteins although the final virus titre was reduced suggesting the interaction was impaired. This work clearly demonstrates that entire domains of lyssavirus G proteins can be exchanged without compromising the function of the G protein itself. Interestingly, substitution of antigenic site III with the corresponding sequence from MOKV did not affect the ability of the G protein to generate infectious particles or to mediate infection but did prevent neutralisation of virus with a monoclonal antibody directed against RABV site III whilst MOKV specific serum neutralised this chimaera. A further study looked at insertion of immunologically potent B and Tc lymphocyte epitopes for other viruses in place of antigenic site III of the Pasteur strain of RABV [52]. Combination the C3 B cell epitope of poliovirus type 1 capsid VP1 and the CTL epitope of lymphocytic choriomeningitis virus (LCMV) nucleoprotein with a truncated G protein induced a weak immunological response in mice but their insertion into a chimeric EBLV1-Pasteur RABV G protein induced both humoral and cellular immune responses against parental lyssaviruses and poliovirus and conferred partial
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J.S. Evans et al. / Vaccine 30 (2012) 7447–7454 Table 2 Amino acid sequence of the lyssavirus antigenic sites. The amino acid sequences are given (numbering after removal of the 19 amino acid signal peptide). The residues coloured in red are those that differ from the RABV sequence, given in the top row.
Virus
Phylogroup
Site II b (34-42)
Site II a (198-200)
Site I (226-231)
Site IV (263-264)
Site III (330-338)
Site 'a' (342-343)
RABV
I
GCTNLSEFS
KRA
KLCGVL
FH
KSVRTWNEI
KG
ABLV
I
GCTSLSGFS
KKA
KLCGIS
FN
KSVRTWDEI
KG
ARAV
I
GCTNLSGFT
KKA
KLCGV M
FH
KSVREWTEV
KG
BBLV
I
GCTTLTVFS
KKA
KLCGVS
FH
KSIRQWTEI
KG
DUVV
I
GCTTLTPFS
KKA
RLCGIS
FH
KSVREWKEI
KG
EBLV-1
I
GCTTLTPFS
KKA
RLCGVP
FH
KSVREWKEV
KG
EBLV-2
I
GCTTLTVFS
KKA
KLCGIS
FH
KSIREWTDV
KG
IRKV
I
GCTTLTAFN
KKA
KLCGMA
DR
KSIREWKEI
KG
KHUV
I
GCTTLSGFT
KRA
KLCGVS
FH
KSIREWSEI
KG
LBV
II
GCSDTATFS
KKS
TLCGKP
NR
LRVDSWNDI
KG
MOKV
II
GCNTESPFT
QKA
TLCGKP
DR
KRVDRWADI
KG
SHIV
II
GCSSSSTFS
KKS
TLCGKP
NR
KRVDRWEEI
KG
WCBV
III
YCTTEQSIT
KLV
SICGRQ
IK
IKVENWSEV
KG
IKOV
?
GCNEGSKVS
ILL
IICGKS
VK
KSVDNWTDI
PI
protection against LCMV. In vivo studies showed that the chimeric G protein could act as a vehicle to successfully express native poliovirus B cell epitope either alone or in association with the LCMV CD8+ T cell epitope at the cell surface and that the fusion of foreign epitopes to the Pasteur virus G antigenic site III significantly increases production of Th cells that are directed at this region. This study concluded that a truncated chimeric G protein was immunologically potent and could be used to carry larger fragments for vaccination. However, one potential problem with this approach is that following the insertion of a foreign epitope, a reduction in RABV-specific neutralising antibodies was seen although the response was still robust enough to enable protection following intracranial challenge [52]. Alongside this work, other studies have more conclusively highlighted the benefit of swapping domains within the G protein between different lyssaviruses to produce a cross-phylogroup antibody response. Jallet et al. [53] generated chimeric G proteins containing domain swaps between RABV and EBLV-1 or MOKV and assessed their potential as DNA vaccines against different lyssavirus species. Here, chimaeras were able to efficiently induce IL-2
Table 3 Human deaths following exposure to lyssaviruses. Virus
Origin
Country
DUVV MOKV MOKV EBLV-1? EBLV-1 EBLV-2 ABLV ABLV EBLV-2 DUVV DUVV IRKV
Bat Unknown Unknown Bat Bat Bat Bat Bat Bat Bat Bat Bat
South Africa Nigeria Nigeria Ukraine Russia Finland Australia Australia Scotland South Africa Kenya Siberia
Human fatality Gender
Age
Male Female Female Female Female Male Female Female Male Male Female Female
31 3.5 6 15 11 30 39 37 55 77 34 ?
Year
References
1970 1969 1971 1977 1985 1985 1996 1998 2002 2006 2007 2007
[62] [32] [63] [64] [65] [19,66] [23] [24,67] [20] [68] [69] [70,28]
producing cells, although the T cell response was greater following stimulation with inactivated homologous virus rather than with the heterologous counterparts. Additionally, the ability of a chimeric G protein containing the site II domain of EBLV-1 and the site III domain of the RABV Pasteur strain was investigated and demonstrated that potent virus neutralising antibodies were generated against both parental viruses. Importantly, in vivo studies showed that the RABV/EBLV-1 chimaera described above was more efficient in its ability to protect mice following intracranial challenge with RABV, EBLV-1 and EBLV-2 than the homogeneous plasmid making it a promising DNA based vaccine candidate. Following on from this approach, Genz et al. [54] generated chimeric rabies vaccines to compare the functionality of different lyssavirus ectodomains with heterologous viral proteins. This experiment established an in vitro and in vivo comparison of G protein by generating recombinant RABV that expressed chimeric G proteins with an internal cytoplasmic domain derived from the parental virus and transmembrane and ectodomains derived from a pathogenic RABV, challenge virus standard (CVS) and also EBLV-1 and 2. The successful recovery and in vitro growth of these viruses demonstrated the flexibility of the rescue system and the recombinant viruses were shown to replicate to comparable virus titres suggesting that the substitution of G protein domains from different lyssavirus species did not affect function. Each of the recombinant viruses was able to target primary neurons which are essential for neuroinvasion and pathogenicity. The conclusions from this study emphasise the potential use of recombinant rabies viruses as suitable pan-lyssavirus vaccine candidates [54]. A further successful reverse genetics strategy was developed by Faber et al. [55] where a recombinant form of a rabies virus vaccine was constructed that contained multiple copies of a G gene from an attenuated virus. This recombinant virus demonstrated that an attenuated G protein expressed in duplicate was able to protect against intra cranial challenge with RABV with a greater level of survivorship than a virus expressing a single G protein. This over expression of G may provide extra protection against challenge through a combination of an increase in the induction of apoptosis due to the greater immunogenicity of two glycoproteins and an
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increase in interferon levels as a result of the larger genome size [55]. This work was expanded on and it has been shown that a recombinant virus expressing an attenuated G in triplicate when given in a single dose is sufficient to provide complete protection against RABV [56]. This leads naturally to the suggestion that construction of a recombinant vaccine virus expressing multiple G proteins, each derived from representatives of each lyssavirus phylogroup would be a suitable mechanism of developing a panlyssavirus vaccine. Studies aimed at developing novel vaccines for companion animals also have implications for the development of novel human vaccines. Following the demonstration that domestic animals vaccinated with standard vaccines were not protected against challenge with phylogroup II viruses [48], novel subunit vaccine formulations were tested using vaccinia virus (vv) as a vehicle to express different lyssavirus G proteins [57]. Several vv recombinants were produced including those expressing WCBV G (vv-WG) or MOKV G (vv-MG) alone, two copies of the RABV G (vv-RGRG), a copy of RABV and MOKV G (vv-RGMG), or a copy of RABV G and the WCBV G (vv-RGWG). Importantly, both the single and double G protein expressing viruses were able to protect against intracranial challenge with homologous virus however protection was only afforded against each challenge virus where the vaccine included the homologous G protein. Interestingly, sera from vv-RGMG or vv-MG vaccinated animals showed significant cross-neutralisation activity against LBV. Despite the clear potential of these novel vaccine formulations, there are safety concerns regarding the use of replicationcompetent lyssaviruses and vaccinia virus based recombinants for pre-immunisation. Ultimately, safety fears render the use of such vaccines unlikely. However, as currently licensed rabies vaccines comprise inactivated, adjuvanted rabies viruses there is a potential for the development of novel pan-lyssavirus vaccines being generated from inactivated preparations of viruses from each phylogroup. This approach may provide protection against all lyssaviruses by generating a cross protective antibody response.
7. Conclusions The continued discovery of novel lyssaviruses, particularly those that are highly divergent from the phylogroup I lyssaviruses is a concern for public health, especially amongst those at occupational risk from infection [1,58]. Clearly, the current rabies vaccines licensed for human and animal use are unable to protect against viruses characterised within phylogroups II or III and consequently novel vaccines are required [1,58]. Whilst the current burden and future threat of human infection with divergent lyssaviruses is not easily quantified, the development of novel discriminatory diagnostic assays for use in the field may improve our understanding of the impact of such viruses and their toll on human life in areas where they are known to be epizootic within different sylvatic animal populations. Regardless of the threat from wildlife, the development of novel vaccines that stimulate a pan-lyssavirus neutralising immune response is of importance to those at occupational risk from infection. Certainly, within the scientific community, cases of laboratory exposures to other viral agents for which there are no available vaccines have again highlighted this need [59–61]. The importance of development of such protective tools cannot be understated.
Conflict of interest All authors declare no conflict of interest.
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Review
Rabies virus vaccines: Is there a need for a pan-lyssavirus vaccine? Jennifer S. Evans a,b , Daniel L. Horton a , Andrew J. Easton b , Anthony R. Fooks a,c , Ashley C. Banyard a,∗ a
Animal Health and Veterinary Laboratories Agency, Woodham Lane, Weybridge, Surrey, KT15 3NB, United Kingdom University of Warwick, Gibbet Hill Road, Coventry, West Midlands, CV4 7AL, United Kingdom c National Consortium for Zoonosis Research, University of Liverpool, Leahurst, Neston, South Wirral, CH64 7TE, United Kingdom b
a r t i c l e
i n f o
Article history: Received 14 August 2012 Received in revised form 5 October 2012 Accepted 7 October 2012 Available online 19 October 2012 Keywords: Lyssaviruses Glycoprotein Rabies Vaccines Protection
a b s t r a c t All members of the lyssavirus genus are capable of causing disease that invariably results in death following the development of clinical symptoms. The recent detection of several novel lyssavirus species across the globe, in different animal species, has demonstrated that the lyssavirus genus contains a greater degree of genetic and antigenic variation than previously suspected. The divergence of species within the genus has led to a differentiation of lyssavirus isolates based on both antigenic and genetic data into two, and potentially a third phylogroup. Critically, from both a human and animal health perspective, current rabies vaccines appear able to protect against lyssaviruses classified within phylogroup I. However no protection is afforded against phylogroup II viruses or other more divergent viruses. Here we review current knowledge regarding the diversity and antigenicity of the lyssavirus glycoprotein. We review the degree of cross protection afforded by rabies vaccines, the genetic and antigenic divergence of the lyssaviruses and potential mechanisms for the development of novel lyssavirus vaccines for use in areas where divergent lyssaviruses are known to circulate, as well as for use by those at occupational risk from these pathogens. Crown Copyright © 2012 Published by Elsevier Ltd. All rights reserved.
Contents 1. 2. 3. 4. 5. 6. 7.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7447 Genetic divergence within the lyssavirus genus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7448 Diagnostic evaluation of lyssavirus infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7450 Genetic and antigenic relationships within the lyssavirus genus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7450 Human rabies vaccines and cross protection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7451 Novel experimental vaccine formulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7451 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7453 Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7453 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7453
1. Introduction Rabies virus (RABV) is a member of the lyssavirus genus, a group of zoonotic pathogens within the family Rhabdoviridae that are of global importance as the cause of a fatal encephalitic disease. RABV is the archetypal lyssavirus and historically is one of the most feared viruses known to man and the only virus that is invariably fatal following the development of clinical disease [1,2]. The genome organisation of members of the lyssavirus genus is relatively simple, encoding just five genes that are transcribed from the non-segmented negative strand RNA genome and translated to produce five viral proteins: the nucleocapsid protein
∗ Corresponding author. Tel.: +44 1932 357722. E-mail address: [email protected] (A.C. Banyard).
(N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and the polymerase protein (L) [3]. The lyssavirus G protein is responsible for attachment and entry into host cells and is considered to be a major determinant of pathogenicity [4–6]. Rabies virus is the most extensively characterised virus within the lyssavirus genus and circulates in volant and non-volant mammalian populations across both the developing and developed world. It is estimated that RABV causes between 24,000 and 93,000 human deaths annually [7] although this figure is considered to be an underestimate [8,9]. Critically, the majority of human deaths attributed to lyssavirus infection occur in the developing world where the virus continues to circulate within dog populations [7,10], with the highest disease burden being in the Indian subcontinent and Africa [11]. While both pre- and post exposure prophylaxis tools have been available to prevent disease caused by RABV for over 30 years these are often unavailable in areas
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Fig. 1. Bayesian phylogenetic analysis of glycoprotein ectodomain sequences from representative lyssavirus species, implemented in BEAST (v1.4.8). The GTR+I model of nucleotide substitution and a chain length of 5 million were used. The maximum clade credibility tree was chosen using Treeanotator (v1.4.8) and viewed using Figtree (v1.1.2) estimated samples sizes were >100 and posterior probabilities at key nodes are shown. Scale bar represents substitutions per site. Accession numbers for each of the lyssavirus species analysed in this study are: ABLV AF801020, RABV CVS-11 EU352767, RABV Pitman Moore AJ871962, RABV Flury-HEP GU565704, BBLV JF311903, KHUV EF614261, EBLV-2 EF 157977, ARAV EF614259, DUVV EU293119, EBLV-1 EU293109 and EU293120, IRKV EF614260, MOKV HM623780 and EU293118, SHIBV GU170201, LBV ‘B’ EF547431, LBV ‘C’ EF547425, LBV ‘D’ GU170202, LBV ‘A’ EF547432, WCBV EF614258 and IKOV JN800509.
where the virus is endemic. There are a suspected 10 million potentially infectious contacts between humans and rabid animals each year which makes rabies an important target for prevention and control strategies [12]. Alongside infection attributed to RABV, other members of the lyssavirus genus are able to cause a fatal encephalitic disease in humans. The majority of lyssavirus species have been isolated from bat populations and whilst infection with RABV is documented, the true burden due to the other lyssaviruses remains largely undefined within both human and animal populations, notably due to a lack of diagnostic assays that are able to differentiate between lyssavirus species [13]. Here we review the current status of rabies vaccines, the protection they afford against different lyssavirus species and the potential burden of other lyssaviruses on animal and human health. Finally, we comment on the need for pan-lyssavirus vaccines to protect both those at risk through local endemnicity of divergent viruses in different populations as well as those at risk through occupational activities. 2. Genetic divergence within the lyssavirus genus The lyssavirus genus is divided into 12 species, the majority of which are predominantly associated with infection in bats (Fig. 1). Within Europe, alongside RABV in terrestrial wildlife populations three other lyssavirus species have been genetically characterised: European Bat Lyssavirus type-1 (EBLV-1), EBLV-2 and Bokeloh Bat Lyssavirus (BBLV). EBLV-1 is further genetically separated into two lineages which show an overlap in geographical incidence. EBLV-1a is found predominantly throughout northern Europe whilst EBLV1b has been detected in southern Germany, France and Spain. Both EBLV-1 lineages are associated with infection of Eptesicus fuscus, the Serotine bat although infection of other bat species such as the Meridional Serotine (E. isabellinus) has suggested the circulation of a third lineage of EBLV-1 [14,15]. EBLV-1 cross species transmission (CST) events have occurred at a very low frequency but with no maintenance of infection within the new host [16–18]. In contrast, EBLV-2 is predominantly associated with infection of
Myotis daubentonii, the Daubenton’s bat, across much of northern Europe and no cross species transmission events have been reported although two fatal human cases have occurred [19,20]. There has been only a single isolate of BBLV, obtained from a Natterer’s bat (Myotis natterei) in Germany in November 2009 [21]. In other parts of the world several other, predominantly bat associated lyssaviruses occur [15]. In Australia, Australian bat lyssavirus (ABLV) has been isolated from five different bat species since its initial isolation in 1996 [22] and has also been implicated in human fatalities [23,24]. A further four Eurasian lyssaviruses have been described, predominantly from single virus isolates from different bat species. These include Aravan virus (ARAV) isolated from a bat in Kyrgystan [25], Khujand virus (KHUV) isolated in Tajikistan [26], Irkut virus (IRKV) isolated from a bat in Eastern Siberia [25] and West Caucasian Bat virus (WCBV) isolated from a bat in the Caucasus mountains in Russia [25]. Phylogenetic classification of these Eurasian isolates have shown them to be distinct species within the lyssavirus genus with WCBV being the most genetically divergent [27]. Of these, only Irkut virus has been associated with human fatality [28]. Across Africa a number of bat lyssaviruses have been detected. The most frequently reported is Lagos bat virus (LBV) which has been isolated from multiple bat species distributed across subSaharan Africa [29]. There are currently four defined lineages of LBV; lineage A consists of Kenyan (2007), Senegalese (1985) and French (1999) isolates, lineage B contains only the original Nigerian isolate, lineage C contains isolates from Zimbabwe, South Africa and the Central African Republic and lineage D contains a single isolate identified in an Egyptian fruit bat in Kenya [30,31]. It has been suggested that Lagos bat virus isolates could be subdivided into several genotypes based on criteria adopted by the ICTV whereby all isolates belonging to the same species have identities of greater than 80%. LBV isolates are more divergent than this across each lineage (Fig. 1) and as such may ultimately be re-classified. Duvenhage virus (DUVV) was initially isolated from a fatal human infection following a bat bite and was subsequently isolated from a number of bat species, mainly Nycteris species in South Africa
Table 1 Nucleotide and amino acid identity of the lyssavirus G protein. Percentage identities are shown. Where identity is less than or equal to 60% they are shaded green. Viruses that have been tentatively classified into phylogroups are shaded accordingly.
Amino acid (% identity) ABLV
LBVD
LBVB
WCBV
48
52
58
56
58
48
50
47
47
53
52 81
LBVC
RABV RV61
DUVV
EBLV 1
73
75
71
73
72
47
48
46
48
48
50
50
50
50
51
77
57
57
57
58
74
55
56
57
59
79
73
57
58
56
80
74
56
58
57
72
59
59
58
58
58 90
SHIBV
MOKV
58
60
58
48
49
48
54
52
53
53
80
82
78
79
81
77
85
IKOV
52
WCBV
55
55
LBVA
59
53
56
LBVF
59
52
58
73
LBVB
59
53
57
73
73
LBVC
58
54
58
72
74
77
SHIBV
60
55
58
71
71
72
72
RABV CVS
MOKV
57
52
57
69
71
69
67
67
RABV CVS
70
53
56
58
59
58
59
60
58
RABV RV61
71
53
54
59
59
60
60
61
59
87
DUVV
67
52
58
58
60
58
58
59
59
65
65
EBLV 1
68
53
57
60
62
59
60
62
61
66
68
BBLV
EBLV 2
KHUV
76
76
76
77
46
47
48
47
51
52
51
51
59
56
57
56
57
58
57
57
58
57
57
59
58
56
57
57
58
59
58
57
57
57
59
58
58
59
57
59
57
58
58
57
57
57
57
69
70
68
73
75
73
75
71
73
69
75
77
76
78
80
75
78
78
79
77
80
84
78
80
79
78
75
76
76
83
85
83
87
86
73
IRKV
ARAV
IRKV
66
53
57
59
60
60
61
60
60
66
67
70
74
ARAV
70
53
57
58
61
60
60
61
60
70
69
70
74
70
BBLV
71
54
58
58
60
60
60
60
60
70
70
71
72
70
74
EBLV 2
71
53
57
58
61
61
61
60
60
70
71
72
74
70
76
77
KHUV
73
53
57
58
60
60
59
59
60
69
70
71
72
75
75
76
J.S. Evans et al. / Vaccine 30 (2012) 7447–7454
Nucleotide (% identity)
ABLV
LBVA
IKOV
88 79
7449
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and Zimbabwe. There have been two further cases of DUVV infection in humans, both of African origin. Shimoni bat virus (SHIBV) was first isolated in 2009 from a Commerson’s leaf-nosed bat in Kenya [31]. Phylogenetically, SHIBV lies between Mokola virus and WCBV (Fig. 1). Mokola virus (MOKV) is an African lyssavirus which is only one of two lyssavirus species that has never been detected within bat species. MOKV has been isolated from a range of mammals including a shrew, domestic cats and a dog. However, sera from LBV positive bats has been shown to neutralise MOKV making assessment of the range of species infected with MOKV difficult and as a result bats cannot be ruled out as a reservoir for MOKV. There have been two recorded human cases of MOKV infection [32]. The latest lyssavirus to be identified is Ikoma lyssavirus (IKOV). IKOV was isolated from an African civet and is only the second non-rabies lyssavirus to have been isolated only from terrestrial carnivore species although its origin within chiropteran species is assumed from the circumstances within which it was detected [33]. IKOV is genetically divergent from all the other lyssaviruses [34]. 3. Diagnostic evaluation of lyssavirus infection Reporting of RABV infection remains problematic across areas of the developing world where the disease is endemic and as a result the estimated annual death toll is believed to be an underestimate of the true burden of disease. However, where virus infection is detected, the current diagnostic assays are unable to differentiate between RABV and the other lyssaviruses. Currently, the ‘Gold Standard’ OIE prescribed test for lyssavirus diagnosis is the fluorescent antibody test (FAT) [35]. This test is only performed on post mortem tissues as it requires a brain smear with polyclonal antibodies used to detect virus antigen. Unfortunately, whilst the antibodies used in the FAT are able to detect all lyssaviruses isolated, they are unable to differentiate between them. Indeed, the most recently identified lyssavirus, IKOV, was characterised following RT-PCR and sequence analysis of antigen positive brain material from terrestrial carnivores in Tanzania. Without the application of genetic characterisation this lyssavirus species would not have been discovered [33]. There have been very few reports describing isolates from human or animal fatalities that have been genetically typed as being caused by lyssaviruses other than RABV. Where this has occurred, initial diagnostic tools have merely confirmed infection with a lyssavirus. Importantly, for many of these infections, even if pre-immunisation of the individual had occurred, depending on the infecting virus, a protective antibody titre may not have been generated. 4. Genetic and antigenic relationships within the lyssavirus genus Across the lyssavirus genus, viruses are classified according to both genetic and antigenic data. Recently, there has been a shift in nomenclature from the definition of lyssavirus genotypes to that of lyssavirus species [36]. In addition to the separation of genetically distinct viruses into species, these viruses can also be divided into phylogroups using antigenic data [36,37]. This differentiation is mainly based on the degree of cross protection afforded by the current rabies vaccines between each of the viruses characterised. Genetic data allow reliable quantitative comparison of lyssavirus divergence [38] and sequence data are available for at least one isolate from all 14 virus species either already classified within the lyssavirus genus or awaiting classification. Across the open reading frame (ORF) encoding the G protein the lyssaviruses are at most 50% divergent at the nucleotide level while sharing only 46% identity at the amino acid level (Table 1). Genetically, IKOV is the most divergent lyssavirus [33]. However, using genetic data to predict
Fig. 2. Schematic representation of the lyssavirus G protein. Defined antigenic sites and conserved cysteine residues are shown. C residues are coloured according to proposed linkages within the mature protein [71]. TM: transmembrane; NH2: amino terminus; COOH: Carboxy-terminus.
vaccine cross protection is limited by the unpredictable effect of amino acid substitutions on antigenic variation, depending on their nature and location. Measuring antigenic variation directly typically relies on less reliable serological assays, which are affected by individual variation in antibody response. Consequently, serological data are considered to have low resolution, and are often only used qualitatively. The improved resolution of antigenic maps generated using antigenic cartography linked to serological assay data has allowed more precise comparison of antigenic and genetic data for pathogens including influenza viruses [38]. Application of these antigenic cartography techniques to a global panel of lyssaviruses has allowed integration of genetic and antigenic data [27]. On average a 4.8% change in the G protein ectodomain amino acid sequence caused a change of one antigenic unit between viruses (equivalent to a two-fold change in antibody titre). Although there is a good correlation (95% CI for r = 0.81–0.88) between genetic distance in the glycoprotein and antigenic distance, over 30% of antigenic variation could not be predicted by genetic distance alone. Investigation of discrepancies between genetic and antigenic distances showed, for example, that IRKV and ARAV are antigenically more closely related to EBLV-2 than EBLV-1, and that KHUV is closer to classical RABV than would be expected from
J.S. Evans et al. / Vaccine 30 (2012) 7447–7454
phylogenetic relationships. LBV and MOKV are antigenically distant and have been placed in a different phylogroup based on genetic distance and limited cross neutralisation [27,39] (Fig. 1). There is a low degree of cross neutralisation between phylogroups I and II and also between both phylogroups and WBCV [27]. In vivo vaccine challenge experiments have shown reduced or no efficacy of current licensed rabies vaccines against viruses in phylogroup II (MOKV, LBV, SHIBV) [40] and phylogroup III (WCBV) [41]. There is also evidence for variable vaccine efficacy against some viruses in phylogroup I [41,42] which suggests a gradual loss of vaccine protection corresponding to antigenic distance from vaccine strains. Improving prediction of vaccine protection using glycoprotein sequence alone would be a significant advance for future vaccine development and also for assessing the possible threat posed by novel lyssaviruses. However the improved accuracy requires more detailed knowledge of the individual antigenic effects of amino acid substitutions. Estimates of the individual antigenic effect of substitutions can be made for example, using known antigenic sites, protein structure and amino acid properties. It is now possible to test those estimates using site directed mutagenesis and antigenic cartography. The lyssavirus G protein which is present on the surface of the virion is the major viral component responsible for induction of a host antibody response and is the target of host neutralising antibodies [43]. The regions of the G protein responsible for the differences in antigenicity seen within what is otherwise a rather conserved genus are ill defined. The G protein is relatively conserved in length across the genus with an ORF encoding between 523 and 534 amino acids. Furthermore all lyssavirus proteins contain 14 highly conserved cysteine residues alongside the antigenic domains that contribute to the structure of the protein. Currently, defined antigenic domains within the G protein include four major antigenic sites and one minor antigenic site (Fig. 2). Historically, these have been defined through scanning mutagenesis experiments and mapping of monoclonal antibody (mAb) binding sites [44]. The positions and a consensus amino acid sequence for each antigenic site are detailed in Table 2. Antigenic site I is a region containing both conformational and linear epitopes as it is delineated by the monoclonal antibody mAb 509–6 that recognises a conformational epitope as well as antibody CR57 that recognises a linear epitope [45]. Antigenic site II has two domains, IIa and IIb so is a discontinuous conformational epitope. Antigenic site III is continuous, though no site specific mAbs are able to bind unfolded protein which indicates that this epitope forms part of a loop on the protein surface and it is this tertiary structure which enables recognition by antibodies or of neuronal receptors [45]. Antigenic site IV consists of only two amino acids and is continuous though it contains overlapping linear epitopes. Minor site ‘a’ is located in close proximity to site III but contains no overlapping epitopes and consists of only two amino acids [45].
5. Human rabies vaccines and cross protection From a public health perspective, the efficacy of rabies vaccines against infection with other lyssaviruses is of great interest to both the scientific and human health communities [1]. All rabies vaccines licensed for use within the developed world consist of inactivated preparations of live attenuated classical rabies virus strains. The provision of protection against the African and Eurasian lyssaviruses remains a problem. Human fatalities have highlighted occasions where death can be attributed to a non-RABV lyssavirus (Table 3). Vaccination and challenge studies in animal models suggest that rabies vaccines provide protection against
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DUVV [42], the EBLVs [46] and some of the recently identified Asian lyssaviruses [41]. However, LBV infection in rabies vaccinated companion animals has highlighted the lack of protection against non-rabies lyssaviruses following vaccination [47,48]. Furthermore, pre- and post-exposure vaccination failed to prevent disease and death in an animal model of WCBV infection [41]. These factors suggest that more cross reactive vaccine formulations may be necessary in areas where a threat to the human population comes from non-rabies lyssaviruses. Recent advances in the antigenic characterisation of different lyssaviruses may also aid future cross-reactive vaccine design [27]. The level of cross protection against IKOV is unknown although sequence comparisons suggest that there will be little or no cross protection afforded by current rabies vaccines against this novel lyssavirus [32]. Few human cases involving infection with non-rabies lyssaviruses have been reported although this may be due to the lack of discriminatory diagnostic tools without which the true burden remains unknown [49,50].
6. Novel experimental vaccine formulations The question of defining protection afforded against different lyssaviruses has been the basis for a number of studies into the development of recombinant viruses or glycoproteins as potential novel vaccine strategies. All approaches to date have focussed on the generation of vaccines that protect against phylogroup I and phylogroup II viruses [1,39]. Mebatsion et al. [51] used a reverse genetics system to generate recombinant viruses containing both heterologous and chimeric lyssavirus G proteins and, using reporter gene expression, showed that these could assemble and function as infectious virions. This study used a MOKV G protein as a heterologous replacement for the wildtype G protein in a RABV full length clone. The G gene of MOKV encodes a 523 amino acid protein, 2 amino acids shorter than that present in RABV with only 56.7% amino acid identity. No sequence homology was identified between the signal sequences or transmembrane and cytoplasmic domains of MOKV and RABV. However, the ectodomains of these two viruses (amino acids 19–430) had 62% amino acid identity although the five defined antigenic sites are poorly conserved (Table 2). Despite this, from a structural perspective all 14 cysteine residues were conserved between these viruses suggesting a similarity at the structural level. Expressing the MOKV G in the RABV full length clone allowed virus rescue and growth. Also, infectious virus was recovered when a chimeric G protein containing the ectodomain or a short surface epitope was substituted for the wildtype RABV G protein. This demonstrated that the cytoplasmic domain of MOKV was able to interact heterotypically with the wildtype RABV proteins although the final virus titre was reduced suggesting the interaction was impaired. This work clearly demonstrates that entire domains of lyssavirus G proteins can be exchanged without compromising the function of the G protein itself. Interestingly, substitution of antigenic site III with the corresponding sequence from MOKV did not affect the ability of the G protein to generate infectious particles or to mediate infection but did prevent neutralisation of virus with a monoclonal antibody directed against RABV site III whilst MOKV specific serum neutralised this chimaera. A further study looked at insertion of immunologically potent B and Tc lymphocyte epitopes for other viruses in place of antigenic site III of the Pasteur strain of RABV [52]. Combination the C3 B cell epitope of poliovirus type 1 capsid VP1 and the CTL epitope of lymphocytic choriomeningitis virus (LCMV) nucleoprotein with a truncated G protein induced a weak immunological response in mice but their insertion into a chimeric EBLV1-Pasteur RABV G protein induced both humoral and cellular immune responses against parental lyssaviruses and poliovirus and conferred partial
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J.S. Evans et al. / Vaccine 30 (2012) 7447–7454 Table 2 Amino acid sequence of the lyssavirus antigenic sites. The amino acid sequences are given (numbering after removal of the 19 amino acid signal peptide). The residues coloured in red are those that differ from the RABV sequence, given in the top row.
Virus
Phylogroup
Site II b (34-42)
Site II a (198-200)
Site I (226-231)
Site IV (263-264)
Site III (330-338)
Site 'a' (342-343)
RABV
I
GCTNLSEFS
KRA
KLCGVL
FH
KSVRTWNEI
KG
ABLV
I
GCTSLSGFS
KKA
KLCGIS
FN
KSVRTWDEI
KG
ARAV
I
GCTNLSGFT
KKA
KLCGV M
FH
KSVREWTEV
KG
BBLV
I
GCTTLTVFS
KKA
KLCGVS
FH
KSIRQWTEI
KG
DUVV
I
GCTTLTPFS
KKA
RLCGIS
FH
KSVREWKEI
KG
EBLV-1
I
GCTTLTPFS
KKA
RLCGVP
FH
KSVREWKEV
KG
EBLV-2
I
GCTTLTVFS
KKA
KLCGIS
FH
KSIREWTDV
KG
IRKV
I
GCTTLTAFN
KKA
KLCGMA
DR
KSIREWKEI
KG
KHUV
I
GCTTLSGFT
KRA
KLCGVS
FH
KSIREWSEI
KG
LBV
II
GCSDTATFS
KKS
TLCGKP
NR
LRVDSWNDI
KG
MOKV
II
GCNTESPFT
QKA
TLCGKP
DR
KRVDRWADI
KG
SHIV
II
GCSSSSTFS
KKS
TLCGKP
NR
KRVDRWEEI
KG
WCBV
III
YCTTEQSIT
KLV
SICGRQ
IK
IKVENWSEV
KG
IKOV
?
GCNEGSKVS
ILL
IICGKS
VK
KSVDNWTDI
PI
protection against LCMV. In vivo studies showed that the chimeric G protein could act as a vehicle to successfully express native poliovirus B cell epitope either alone or in association with the LCMV CD8+ T cell epitope at the cell surface and that the fusion of foreign epitopes to the Pasteur virus G antigenic site III significantly increases production of Th cells that are directed at this region. This study concluded that a truncated chimeric G protein was immunologically potent and could be used to carry larger fragments for vaccination. However, one potential problem with this approach is that following the insertion of a foreign epitope, a reduction in RABV-specific neutralising antibodies was seen although the response was still robust enough to enable protection following intracranial challenge [52]. Alongside this work, other studies have more conclusively highlighted the benefit of swapping domains within the G protein between different lyssaviruses to produce a cross-phylogroup antibody response. Jallet et al. [53] generated chimeric G proteins containing domain swaps between RABV and EBLV-1 or MOKV and assessed their potential as DNA vaccines against different lyssavirus species. Here, chimaeras were able to efficiently induce IL-2
Table 3 Human deaths following exposure to lyssaviruses. Virus
Origin
Country
DUVV MOKV MOKV EBLV-1? EBLV-1 EBLV-2 ABLV ABLV EBLV-2 DUVV DUVV IRKV
Bat Unknown Unknown Bat Bat Bat Bat Bat Bat Bat Bat Bat
South Africa Nigeria Nigeria Ukraine Russia Finland Australia Australia Scotland South Africa Kenya Siberia
Human fatality Gender
Age
Male Female Female Female Female Male Female Female Male Male Female Female
31 3.5 6 15 11 30 39 37 55 77 34 ?
Year
References
1970 1969 1971 1977 1985 1985 1996 1998 2002 2006 2007 2007
[62] [32] [63] [64] [65] [19,66] [23] [24,67] [20] [68] [69] [70,28]
producing cells, although the T cell response was greater following stimulation with inactivated homologous virus rather than with the heterologous counterparts. Additionally, the ability of a chimeric G protein containing the site II domain of EBLV-1 and the site III domain of the RABV Pasteur strain was investigated and demonstrated that potent virus neutralising antibodies were generated against both parental viruses. Importantly, in vivo studies showed that the RABV/EBLV-1 chimaera described above was more efficient in its ability to protect mice following intracranial challenge with RABV, EBLV-1 and EBLV-2 than the homogeneous plasmid making it a promising DNA based vaccine candidate. Following on from this approach, Genz et al. [54] generated chimeric rabies vaccines to compare the functionality of different lyssavirus ectodomains with heterologous viral proteins. This experiment established an in vitro and in vivo comparison of G protein by generating recombinant RABV that expressed chimeric G proteins with an internal cytoplasmic domain derived from the parental virus and transmembrane and ectodomains derived from a pathogenic RABV, challenge virus standard (CVS) and also EBLV-1 and 2. The successful recovery and in vitro growth of these viruses demonstrated the flexibility of the rescue system and the recombinant viruses were shown to replicate to comparable virus titres suggesting that the substitution of G protein domains from different lyssavirus species did not affect function. Each of the recombinant viruses was able to target primary neurons which are essential for neuroinvasion and pathogenicity. The conclusions from this study emphasise the potential use of recombinant rabies viruses as suitable pan-lyssavirus vaccine candidates [54]. A further successful reverse genetics strategy was developed by Faber et al. [55] where a recombinant form of a rabies virus vaccine was constructed that contained multiple copies of a G gene from an attenuated virus. This recombinant virus demonstrated that an attenuated G protein expressed in duplicate was able to protect against intra cranial challenge with RABV with a greater level of survivorship than a virus expressing a single G protein. This over expression of G may provide extra protection against challenge through a combination of an increase in the induction of apoptosis due to the greater immunogenicity of two glycoproteins and an
J.S. Evans et al. / Vaccine 30 (2012) 7447–7454
increase in interferon levels as a result of the larger genome size [55]. This work was expanded on and it has been shown that a recombinant virus expressing an attenuated G in triplicate when given in a single dose is sufficient to provide complete protection against RABV [56]. This leads naturally to the suggestion that construction of a recombinant vaccine virus expressing multiple G proteins, each derived from representatives of each lyssavirus phylogroup would be a suitable mechanism of developing a panlyssavirus vaccine. Studies aimed at developing novel vaccines for companion animals also have implications for the development of novel human vaccines. Following the demonstration that domestic animals vaccinated with standard vaccines were not protected against challenge with phylogroup II viruses [48], novel subunit vaccine formulations were tested using vaccinia virus (vv) as a vehicle to express different lyssavirus G proteins [57]. Several vv recombinants were produced including those expressing WCBV G (vv-WG) or MOKV G (vv-MG) alone, two copies of the RABV G (vv-RGRG), a copy of RABV and MOKV G (vv-RGMG), or a copy of RABV G and the WCBV G (vv-RGWG). Importantly, both the single and double G protein expressing viruses were able to protect against intracranial challenge with homologous virus however protection was only afforded against each challenge virus where the vaccine included the homologous G protein. Interestingly, sera from vv-RGMG or vv-MG vaccinated animals showed significant cross-neutralisation activity against LBV. Despite the clear potential of these novel vaccine formulations, there are safety concerns regarding the use of replicationcompetent lyssaviruses and vaccinia virus based recombinants for pre-immunisation. Ultimately, safety fears render the use of such vaccines unlikely. However, as currently licensed rabies vaccines comprise inactivated, adjuvanted rabies viruses there is a potential for the development of novel pan-lyssavirus vaccines being generated from inactivated preparations of viruses from each phylogroup. This approach may provide protection against all lyssaviruses by generating a cross protective antibody response.
7. Conclusions The continued discovery of novel lyssaviruses, particularly those that are highly divergent from the phylogroup I lyssaviruses is a concern for public health, especially amongst those at occupational risk from infection [1,58]. Clearly, the current rabies vaccines licensed for human and animal use are unable to protect against viruses characterised within phylogroups II or III and consequently novel vaccines are required [1,58]. Whilst the current burden and future threat of human infection with divergent lyssaviruses is not easily quantified, the development of novel discriminatory diagnostic assays for use in the field may improve our understanding of the impact of such viruses and their toll on human life in areas where they are known to be epizootic within different sylvatic animal populations. Regardless of the threat from wildlife, the development of novel vaccines that stimulate a pan-lyssavirus neutralising immune response is of importance to those at occupational risk from infection. Certainly, within the scientific community, cases of laboratory exposures to other viral agents for which there are no available vaccines have again highlighted this need [59–61]. The importance of development of such protective tools cannot be understated.
Conflict of interest All authors declare no conflict of interest.
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