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Vaccine 28 (2010) 3896–3901

Contents lists available at ScienceDirect

Vaccine

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eview

he immune response to rabies virus infection and vaccination

icholas Johnsona,∗, Adam F. Cunninghamb, Anthony R. Fooksa

Rabies and Wildlife Zoonoses Group, Veterinary Laboratories Agency – Weybridge, Woodham Lane, Addlestone, Surrey KT15 3NB, United KingdomMedical Research Council Centre for Immune Regulation, University of Birmingham, Birmingham B15 2TT, United Kingdom

r t i c l e i n f o

rticle history:eceived 15 October 2009eceived in revised form 10 February 2010ccepted 21 March 2010vailable online 3 April 2010

eywords:abies virus

nfectionaccination

mmune responsentigen presentation

a b s t r a c t

Infection with rabies virus causes encephalitis in humans that has a case fatality rate of almost 100%. Thisinability to resolve infection is surprising since both pre-exposure vaccination and, if given promptly,post-exposure vaccination is highly effective at preventing encephalitic disease. The principal immuno-logical correlate of protection produced by vaccination is neutralizing antibody. T-helper cells contributeto the development of immunity whereas cytotoxic T cells do not appear to play a role in protection andmay actually be detrimental to the host. One reason for a failure to protect in humans may be the poorimmunological response the virus provokes, despite the period between exposure to virus and the devel-opment of disease being measured in months. Few individuals have measurable neutralizing antibodyon presentation with disease, although in many cases this develops as symptoms become more severe.Furthermore, when antibody is detected in serum it rarely appears in cerebrospinal fluid suggesting lim-ited penetration into the CNS, the site where it is most needed. The role of the modest mononuclearcell infiltrate into the brain parenchyma is unclear. Some studies suggest the virus can suppress cell-mediated immunity early during the infection although there is little mechanistic evidence to supportthis beyond suppression of intracellular interferon production by the viral phosphoprotein. In contrast,

levels of antibody in the CNS correlate to the peak virus production within the CNS. Here we reviewthe current understanding of immune responses to rabies infection and vaccination against this disease.This article identifies a need to understand how rabies antigens are initially presented and how this caninfluence the subsequent development of antibody responses. This could help identify ways in which theresponse to prophylactic vaccination can be enhanced and how the natural immune response to infection can be boosted to combat neuroinvasion.

Crown Copyright © 2010 Published by Elsevier Ltd. All rights reserved.

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38962. The immune response to vaccination. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38973. The immune response to infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38984. Rabies virus immunosuppression of the host . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38985. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3899

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3900References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3900

. Introductionall have caused or have the potential to cause rabies in man. Thelyssaviruses have a 12 kb genome encoding five proteins [1], in theorder: nucleoprotein (N), phosphoprotein (P), matrix (M), glyco-

Rabies virus (RABV) is the type species of the genus Lyssavirus,ithin the Rhabdoviridae family. The Rhabdoviridae are negative

ense single-stranded RNA viruses with a distinctive bullet-shapetructure. A further 10 viruses belong to the Lyssavirus genus and

∗ Corresponding author. Tel.: +44 1932 357937; fax: +44 1932 357239.E-mail address: n.johnson2@vla.defra.gsi.gov.uk (N. Johnson).

264-410X/$ – see front matter. Crown Copyright © 2010 Published by Elsevier Ltd. All rioi:10.1016/j.vaccine.2010.03.039

protein (G) and RNA-dependent-RNA polymerase (L). RABV causesacute encephalitis in mammals with a case fatality rate of almost100%. The virus persists in endemic cycles within carnivore or chi-ropteran reservoirs and humans are typically exposed through an

animal bite. The majority of human cases occur in Africa and Asiawhere the dog is the principal vector of transmission. The typeof bite varies widely depending on the vector encountered. Dogscan transmit rabies through licking of mucosal surfaces or breaks

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n the skin. More commonly infection is associated with severe,eep bites, especially in children, and in limbs as victims attempto defend themselves. Such bites lead to the deposition of viruseep into muscle tissue. By contrast, bats appear to efficiently trans-it rabies to humans through shallow, intradermal bites, often

oing un-noticed by the victim [2]. Aerosol transmission has beenbserved under extreme conditions [3,4], however this mechanisms unlikely to contribute to natural transmission of the disease. It islso possible that infection in these cases could have resulted fromral exposure.

Rabies virus, like all members of the Lyssavirus genus, is neu-otropic and following a bite infects peripheral nerves close tohe bite site. Virus then moves by retrograde axonal transport tohe dorsal root ganglia where virus replication becomes detectable5]. This movement can be extremely rapid. In murine models ofnfection, amputation or severance of the sciatic nerve followingnoculation in the footpad could prevent the development of dis-ase [6]. However, in experimental models this severe methodf therapy loses its efficacy within days of inoculation. Axoplas-ic transport of rabies virus within cultured rat sensory neurons

eached 12–24 mm/day [7]. This conflicts with disease in humanshere the time period between exposure and the development of

abies can be measured in months, even years in extreme cases.t is assumed that the virus persists, either in a dormant state oreplicates at very low levels possibly in muscle tissue, close to theite site. Rabies virus was detectable by reverse transcriptase poly-erase chain reaction (RT-PCR) for 2 months in tissue close to the

oint of exposure of striped skunks (Mephitis mephitis) inoculatedith lose doses of rabies virus [8]. Immunohistochemical labeling

uggested that this virus was associated with striated muscle fibres.number of cell-surface protein receptors have been identified,

ne being the acetylcholine receptor that is found at nerve–muscleunctions and could enhance the entry of RABV into the peripheralervous system [9].

Once virus has entered the nervous system, movement to therain via the spinal cord is rapid and associated with an explosive

ncrease in virus replication. Initial indications of disease includeain or paraesthesia close to the bite site and are often associatedith fever, fatigue and weakness in associated limbs. Non-specificeurological symptoms including headache and anxiety are oftenxperienced for a number of days before the development of overtncephalitis and it is usually only at this point that patients seekedical help and admission to hospital [10]. Currently no validated

herapy is available once disease symptoms develop and deathnsues within a number of days or weeks dependent on the levelf clinical intervention [11].

Successful treatment of rabies is reliant on responding to anxposure immediately. Firstly, wound cleaning is a highly effectivend a low cost means of reducing the risk of acquiring infection.econdly, appropriate post-exposure prophylaxis (PEP) consistingf vaccination and rabies immunoglobulin (RIG) is effective at pre-enting disease. Modern rabies vaccines consist of inactivated viruserived from continuous cell lines that are given either intramuscu-

arly or intradermally following World Health Organisation (WHO)ecommended protocols. These regimens have been developed tonsure optimal immune response and typically consist of multiplenjections over a period of 4 weeks.

Two fundamental questions are posed by this pathogen. Firstly,hat are the correlates of immunity and secondly why, does the vic-

im fail to produce a protective response that successfully resolveshe infection or reduces the severity of disease? The first has been

nswered in detail but the second still challenges our understand-ng of the underlying pathogenesis and immune response to rabiesirus. This should be assessed with the appreciation that whilstisease is invariably fatal, exposure does not necessarily result inisease. It is possible that there was no actual exposure to virus or

8 (2010) 3896–3901 3897

that exposure occurred but this stimulated an immune responsethat controlled the virus at an early stage. In both scenarios, theseindividuals do not come to the attention of clinicians, much less bestudied, so it is not clear whether a naturally protective responseis possible against rabies. Serosurveys of animals for lyssaviruses,particularly bats, often identify seropositive, healthy individuals[12–15]. The assumption is that they have been exposed to repli-cating virus although it is not clear how this fits with currentmodels of infection. One hypothesis, again for bats, is that this isa consequence of aerosol exposure within caves [16]. Alternativeexplanations include repeated low dose exposure to virus or someform of abortive infection, but there is little in the way of firmevidence to support any of these hypotheses.

2. The immune response to vaccination

Although there have been a small number of cases of survivorsfollowing infection with rabies [17,18], the vast majority of humanswho develop rabies, die as a consequence of infection. Despitethis, vaccination is highly effective at preventing disease whenadministered before or shortly after exposure to virus. Louis Pas-teur pioneered this approach in 1885 originally using desiccatedspinal cords derived from rabies-infected rabbits. Subsequent vac-cines were also derived from neural tissue from a variety of animalsources and were effective and affordable throughout the world.However, the high content of myelin basic protein in this typeof vaccine leads to a small number of cases of fatal encephalitis[19] and because of this, these types of vaccines are not rec-ommended for use. Current vaccines consist of inactivated virusgrown in continuous cell-lines. Two vaccines are licensed in theUK for human use, human-diploid-cell-vaccine (HDCV) manufac-tured by Aventis Pasteur and purified chick-embryo-cell-vaccine(PCECV) manufactured by Chiron. Pre-exposure vaccination, suchas that given to health-care and laboratory workers, and travelers torabies-endemic areas, is administered in three doses intramuscu-larly at 0, 7 and 28 days. Immunoglobulin M (IgM) is detectablewithin 4 days post-inoculation with HDCV with IgG appearingat day 7. Follow-up studies have shown that responses persistfor up to 2 years after vaccination and passive transfer studiessuggest that it is IgG that provides the most effective protectionagainst disease probably because of the inability of IgM to pene-trate tissue [20]. Prompt post-exposure treatment or prophylaxis(PEP) is the only effective treatment for the prevention of rabies.Unlike pre-exposure vaccination, the regimens recommended bythe World Health Organisation (WHO) for PEP consist of inten-sive re-exposure to vaccine over a short period of time. A standardcourse would be repeated intramuscular inoculation at 0, 2, 7, 14and 28 days [17]. Alternative regimens use intradermal inocula-tion [21,22] that can use a lower volume of vaccine and is thusmore cost-effective where resources are limited. This approachis gaining wide acceptance, especially in Asia [23–25]. The rea-sons for improved responses to intradermal vaccination over theintramuscular route are believed to result from improved antigenpresentation from the skin although this explanation has not beendemonstrated experimentally for RABV.

Vaccine potency is a key factor in judging the effectiveness ofRABV vaccines and usually reflects both the antigen content of aparticular batch and the titre of antibody induced following inoc-ulation. The key parameter measured in humans or animals, isthe titre of neutralizing antibody induced following vaccination.

Two neutralization assays are used to measure this, the rapid flu-orescent focus inhibition test (RFFIT) and the fluorescent antibodyneutralization assay (FAVN). The latter is used to measure compan-ion animal sera prior to entry into the UK under the current PETtravel scheme [26]. The RFFIT has been used to monitor the suc-

3898 N. Johnson et al. / Vaccine 28 (2010) 3896–3901

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ig. 1. Detection of rabies neutralizing antibodies in serum (A) and cerebrospinaluid (B). This data was obtained from a recent human rabies case in the Unitedingdom. The X-axis refers to days post-hospitalization. A logarithmic scale haseen used to display the antibody response.

ess of anti-rabies oral baiting schemes targeting fox populationshroughout Europe [27].

. The immune response to infection

The primary correlate of protection, as demonstrated by theffectiveness of post-vaccination to virus challenge and numerousxperimental studies, is the presence of neutralizing antibody. Inxperimental models of infection using gene knock out (k.o.) mice,hose strains that were susceptible to attenuated strains of rabiesirus were those that lacked all T and B cells or only B cells, whilstice only lacking CD8 T cells were not susceptible [28]. Surpris-

ngly, the majority of human rabies cases do not have detectablentibody responses until some days after the development of acuteisease. In a study of rabies admissions in Thailand only 3 of 11ases had detectable neutralizing antibody to the viral glycoprotein29]. Even in these cases the titres were particularly low, varyingrom 0.26 to 3.42 international units per ml serum (IU/ml). In 6 ofhese patients, where cerebrospinal fluid (CSF) was investigated,o anti-rabies antibody was detected. A similar review of humanases in the USA showed that none of the patients had detectableeutralizing antibody against rabies on presentation to hospital,lthough over half seroconverted within 10 days [30]. Of 14 patientsho were investigated for antibody in the CSF, only 2 had measur-

ble levels. This picture of late developing antibody response waslso observed in two human case of rabies in the UK. In the firstf these cases anti-rabies antibody titres only appeared at day 16fter hospital admission [31]. In the second case, the appearance of

ntibodies within both serum and CSF occurred over a week afterdmission to hospital (Fig. 1). A previous case of human rabies inhe UK, infected with a related virus, European bat lyssavirus type, had no detectable anti-viral response at any time during hospi-alization [32]. Of particular interest in recent years has been the

Fig. 2. Induction of neutralizing antibody in response to infection with RABV.Groups of CD1 outbred mice (n = 5) were inoculated in the left-hind footpad with3.99 × 105 TCID50/ml RABV (strain RV61)[65] and sampled at days 0, 5 and 11. Neu-tralizing antibody titres were measured using the FAVN method [66].

case of a human survivor of rabies of bat origin in the USA [18].The patient had detectable antibody in both CSF and serum onadmission and the treatment applied consisted partly of inductionof coma with the express aim of allowing time for the humoralimmune response to develop. High IgG titres in both CSF andserum did develop and it is possible these contributed to the sur-vival of this patient. Two reports from Colombia and Brazil suggestthat there have been further survivors, however, most attempts torepeat this approach to date have been unsuccessful [33]. The vastmajority of cases end in the death of the patient and the absence ofprotective antibody at the site of virus replication is likely to con-tribute to these poor rates of survival. For the serological responseof the patient shown in Fig. 1, seroconversion occurred late duringthe course of the disease, did not reach a particularly high titre inthe CSF and reached a plateau relatively quickly. All these factorsmay have contributed to the unsuccessful outcome for this partic-ular patient. A more rapid response to infection, particularly beforethe virus has entered the brain and the ability of that antibody toenter the CNS would be expected to enhance survival or limit spreadof virus. Passive transfer of monoclonal antibodies that can inhibitcell-to-cell spread of virus has been observed [34] and suggests thatantibodies can contribute to viral clearance from the CNS althoughthe development of such strategies in man have not been successful.

During infection, it is unclear from where the antigen that drivesthe antibody response is derived, either from virus in the periph-ery or from virus in the CNS. Again rodent experimental models ofinfection have severe limitations in understanding natural infec-tion. In a Syrian hamster model of infection with virulent virus,IgG2 antibody was detected as early as 5 days post-challenge [35].However, the virus was injected by the intra-peritoneal route, andefficiently induced rabies within 8 days. We have observed therapid induction of neutralizing antibodies (within 5 days) to foot-pad inoculation [36] (Fig. 2). There have been no studies to definethe isotype of these antibodies and in the serum the earliest Ab islikely to be IgM. A caveat to these studies is that the level of virusused is likely to be considerably higher than that transmitted insaliva. However, such titres of virus are needed to ensure consistentinduction of disease in a majority of the animals inoculated. Fur-ther work is needed to refine these models to better reflect naturalinfection.

4. Rabies virus immunosuppression of the host

The reasons for the host’s limited responsiveness to infectioncould reflect a number of factors. The neurotropism of the virusresults in the vast majority of replication occurring firstly in thedorsal root ganglion and then progressively through the CNS.Traditionally this has been considered an immunoprivileged loca-

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ion and thus not under the same level of immunosurveillance asther organs. Furthermore, the ability of the immune system tonduce inflammation (i.e. encephalitis) is tightly regulated to pre-ent bystander damage that could leave the host with neuronaleficit. This dogma is changing with a better understanding of the

ymphatic drainage of the brain and spinal cord [37], and the iden-ification of cell trafficking within the CNS [38]. However, this aloneannot explain the failure to deal with rabies virus as a number ofther viral infections including Herpes viruses [39] and Borna dis-ase virus (BDV) [40] can infect the brain, and in the case of BDVersistently, yet are effectively controlled by a functional immuneystem.

A second explanation could be that the infectious dose admin-stered during a bite is too small to trigger immune responsesnabling the virus to infect local sensory nerves. This could wellappen in many circumstances. Bats appear to be efficient trans-itters of rabies but the level of virus in saliva is barely measurable

sing RT-PCR methods. It has been suggested that a bat variant ofabies found only in North America could be adapted to transmis-ion at low doses and favour replication at temperatures below7 ◦C [41]. This may better reflect surface skin temperature andhere is increasing evidence that the sub-dermal inoculation routes a more effective means of infecting experimental animals withuropean bat lyssaviruses when directly compared to intramuscu-ar inoculation.

A final explanation for the lack of antibody could result frommmunosuppression induced by the virus. Mechanisms of both sys-emic immunosuppression and immune inhibition at the cellularevel have been proposed. Observations of lymphoid depletion as aesult of rabies virus infection were made in the late 1980s [42,43].his lymphopenia appeared to affect all lymphoid tissue includ-ng the thymus, spleen and lymph nodes and all cell types [44].he cause proposed for this effect was adrenal hormone toxicity ofymphoid tissues, as mice adrenalectomized prior to infection didot suffer similar depletion. Further evidence for immunosuppres-ion was manifest as reduced cell-mediated responses to both theitogen concanavalin A [45] and the rabies virus specific antigen

46]. Recent research has suggested that rather than a hormonalmbalance causing systemic immunosuppression, cytokines maye responsible, and specifically production of tumour necrosis fac-or alpha (TNF-�) in the CNS during infection [47]. This is supportedy the improved survival of TNF-� receptor knock out mice inesponse to infection with neurovirulent rabies virus [48]. How-ver, TNF-� transcription increases in the brain of mice infectedith avirulent strains [49] and we have not observed increases

n serum TNF-� in mice infected with both RABV and EBLV-2VLA, unpublished data). Also, many of these immunological per-urbations are only observed in mice once disease develops. In thebsence of large-scale virus replication and damage to neurons, it isnclear how the immune response could be suppressed, especially

n draining lymph nodes that might encounter antigen shortly afterbiting event. Some have suggested that both lymphocytes [50]

nd macrophages [51] are susceptible to infection, and infection ofhese cells has a profound effect on the cytokine expression profileseen.

A further possible immunosuppressive mechanism withinirally infected cells has been identified. Recent studies have nowemonstrated that in vitro, RABV is able to suppress the actionf interferons [52,53]. This is mediated by the phosphoproteinhat binds STAT isoforms, prevents their accumulation in the cyto-lasm and disrupts interferon signaling. It has also been shown

o inhibit translocation of STAT dimers into the nucleus and ini-iating transcription of interferon and interferon-inducible genes54]. However, numerous studies have shown that inflammatoryytokines are produced within the CNS during infection suggestinghere may be other pathways active in vivo that can compensate for

Fig. 3. T cell staining of rabies virus infected dorsal root ganglia (day 11). Brownstaining shows binding for murine anti-CD3 on formalin fixed sections. A descriptionof methods can be found in Hicks et al. [67].

this suppression [49,55–59]. It is unclear from these in vivo studieswhether the source of CNS cytokines is directly due to RABV infec-tion (i.e. neurons) or is generated by bystander cells or populationsentering the CNS.

5. Conclusions

The immune privileged status of the CNS and the blood–brainbarrier (BBB) could explain some of the delays in the developmentof a protective response. Virus replication only increases once RABVhas entered the CNS so the level of antigen in the periphery is likelyto be limited. Also, although there is now clear evidence for anti-gen presentation in the CNS and rapid drainage from the spine andCNS to local lymphoid tissue [60], this could produce a delay inpresentation of antigen to appropriate B cells and it seems unlikelythat spleen is a relevant site for immune responses to rabies virus.Once B cells are stimulated and the process of maturation beginsthere is then the challenge to deliver antibodies or plasmacytoidcells back into the CNS. Again, experimental models have showninfiltration of both T and B cells into the dorsal root ganglia, spinalcord and brain [36] (Fig. 3). However, the majority of these cellsare T cells, and most appear to be destined to apoptose shortlyafter entering the CNS [56]. Furthermore there are intrinsic com-plexities in enacting immune responses in the CNS. These includetight regulation of MHC expression [61], high levels of Fas-mediatedapoptosis in lymphocytes and the expression by neuronal cells ofimmunosuppressive factors. The BBB remains intact during rabiesvirus infection [62] and would likely exclude antibodies, althoughthis does not appear to be complete as one report has suggestedthat a neutralizing monoclonal antibody can clear virus from theCNS when given intravenously [63]. Variation between virus strainsmay play a role in the rate of spread through the CNS, BBB per-meability and the speed of development of the immune response.A number of studies have suggested that the virus suppressesthe adaptive immune response. There is certainly compelling evi-dence that the viral phosphoprotein inhibits interferon responsesalthough infection is associated with the increase in gene tran-scripts for interferon-inducible genes. It is likely that inhibitionis transitory and provides a short delay in the host response that

gives the virus a “headstart”. However, there is a paradox in thatthe later stages of infection where viral burden is at its highest andencephalitis is at its most severe appears to be the one point wherethe host immune system is at its most active. The suppression ofperipheral immune responses is more controversial as it is not clear

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ow the virus can achieve this in organs such as the spleen where noirus is present and there are no known soluble factors generatedy the virus that could mediate this.

A single article has investigated that presentation of RABV anti-en in association with immune-stimulating complexes [64]. Thistudy showed the uptake of antigen by marginal zone macrophagesn the spleen following i.v. injection and the subsequent develop-

ent of antibody responses in the presence or after depletion ofacrophages by clodronate liposome treatment. There have been

o studies looking at the antigen presentation of rabies virus orcell maturation in response to anti-rabies vaccination. This sug-

ests that rabies virus infection may prove a useful paradigm inhe investigation of all aspects of antibody development and coulddentify the source of infiltrating lymphocytes present during theater stages of infection. It will also have practical benefits in under-tanding the lack of immune development during infection anddentification of strategies that might improve the induction ofmmunity by post-exposure prophylaxis.

cknowledgements

This work has been jointly funded by European Union (Epizoneorkpackage 5.3) and Defra, UK grant SV3500.

eferences

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