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Vaccination prevents latent HSV1 infection of mouse brain
Neurobiology of Aging 22 (2001) 699 –703
www.elsevier.com/locate/neuaging
Open peer commentary
Vaccination prevents latent HSV1 infection of mouse brain Woan-Ru Lina,1, Roy Jenningsb, Trixie L. Smithb, Matthew A. Wozniaka, Ruth F. Itzhakia,* a
University of Manchester Institute of Science & Technology, Molecular Neurobiology Laboratory, Department of Optometry & Neuroscience, Manchester M60 1QD, UK b University of Sheffield Medical School, Division of Molecular & Genetic Medicine, Sheffield S10 2RX, UK Received 26 July 2000; received in revised form 20 October 2000; accepted 22 December 2000
Abstract Herpes simplex encephalitis (HSE) is a rare but very serious disorder caused by herpes simplex type 1 virus (HSV-1). Treatment with acyclovir decreases mortality but many patients still suffer cognitive impairment subsequently. A vaccine against HSV1 would therefore be of great value. HSV-1 has been implicated also in Alzheimer’s disease (AD): we established that HSV1 resides in the brain of about two thirds of AD patients and aged normal people, and that in carriers of the type 4 allele of the apolipoprotein E gene, it is a strong risk factor for AD. Thus a vaccine against HSV-1 might prevent development of AD in some cases. To find whether a vaccine of mixed HSV-1 glycoproteins (ISCOMs), which protects mice from latent HSV-1 infection of sensory ganglia, prevents HSV1 latency in the CNS, ISCOM-vaccinated or unvaccinated animals were infected with HSV-1. Using polymerase chain reaction (PCR) we detected HSV-1 in brain from 16 of 39 unvaccinated mice (41%), but only 3 of 41 vaccinated mice (7%) (P ⬍ 0.001). Thus, ISCOMs protect the CNS also, suggesting their possible future usage in humans. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Herpes simplex type 1 virus; Vaccine; Mice; Brain; Herpes simplex encephalitis; Alzheimer’s disease; PCR
1. Introduction Primary herpes simplex type 1 virus (HSV-1) infection usually occurs during childhood, with infection typically developing as a result of the direct inoculation of infected droplets from orolabial or nasal secretions on to susceptible mucosal surfaces. HSV-1 infection is virtually universal — most adults are seropositive. Following initial infection with HSV-1, the virus establishes latent infection in the trigeminal ganglia (TG), from which the virus reactivates periodically causing recurrent disease, manifested as cold sores (herpes labialis), in certain people [24]. However, there are many individuals with no history of any disorder caused by HSV-1 infection yet who show evidence of prior infection. HSV-1 is responsible also for most cases of herpes simplex encephalitis (HSE) [24], a brain disease which has an annual incidence estimated at between 1 in 250000 and 1 in a million of the population. HSE causes high mortality as well * Corresponding author. Tel.: ⫹00-44-161-200-3879; fax: ⫹00-44161-200-4433. E-mail address: [email protected] (R.F. Itzhaki). 1 Current address: Epilepsy Unit and Neuropathology Laboratory, Institute of Neurology, Queen’s Square, London WC1N 3BG.
as significant morbidity in survivors. Even though treatment of HSE with acyclovir in the early stages of the disease decreases mortality, patients are not free of further complications. Many suffer from severe cognitive impairments which can seriously affect their quality of life. Therefore it is of great importance to develop a vaccine against HSV-1 so that the suffering caused by both herpes labialis and the more serious condition, HSE, is alleviated or even prevented. Our previous studies established that HSV-1 can reside latently within the brains of a high proportion of aged people [22,23]; it has a predilection for the temporal lobe, frontal lobe and hippocampus. Subsequent studies by other groups have confirmed the presence of HSV1 in human brain [2,3,16]. We have since found that the combination of HSV-1 in brain and carriage of the type 4 allele of the apolipoprotein E gene (apoE-4) is a strong risk factor for Alzheimer’s disease (AD), and that neither HSV-1 nor apoE-4 alone is a risk [21,29]. We found also, in striking parallelism in the peripheral nervous system (PNS), that apoE-4 is a risk factor for herpes labialis [21,29]. Another link between HSV1 and AD is the recent finding that a fragment of HSV1 glycoprotein B has sequence homology to the A pepide found in the characteristic plaques of AD brain [8]. As to interactions between viruses and lipopro-
0197-4580/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 1 9 7 - 4 5 8 0 ( 0 1 ) 0 0 2 3 9 - 1
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W.R. Lin et al. / Neurobiology of Aging 22 (2001) 699 –703
teins (or their components), other cases have been described in diverse types of virus (see review, ref. [9]), the most relevant being between apoE-4 and the onset of dementia and peripheral neuropathy in HIV-infected (pre-AIDS) subjects [7]. On the basis of our findings in AD, we suggested that the combination of virus and apoE-4 is particularly harmful in the nervous system and that the virus in brain reactivates periodically, causing more damage — and eventually AD — in apoE-4 carriers. HSV-1 in brain is the first environmental agent to be definitely implicated in AD, and our results strengthen the rationale for developing a vaccine against HSV-1. As to HSV-1 infection of the mouse nervous system, the discovery that administration of HSV-1 at various sites caused acute infection and later, establishment of a latent infection in the PNS, led to usage of mice as a model for reactivation of the virus in the PNS of humans [19,34,36]. Studies on the mouse central nervous system (CNS), using explantation, co-cultivation, or molecular hybridisation, showed that after HSV-1 infection, viral latency could be established in brain also, following acute infection [6,26, 27]; it was therefore suggested that this might be a suitable model for investigating neurological diseases with a known or a possible viral aetiology. More recently, HSV1-infected mice have been used as a model for HSE, seeking the viral DNA and viral transcripts in brain by PCR [10]. Vaccines against HSV-1 have taken several forms over the years. Initially, inactivated vaccines derived from phenol-treated tissues from infected animals were used, but these contained animal proteins and their administration led to demyelination; hence this procedure was not pursued further. Subsequently, ultraviolet light-inactivated purified viruses derived from tissue culture were used. However, the vaccine was not effective and sometimes caused side effects. In more recent years, replication-defective HSV-1 vaccines [30,32,35], vectored HSV glycoproteins-usually gD or gB [14,25,37], DNA vaccines [28,31] and adjuvanted glycoprotein vaccine preparations [4,5] have been assessed in animal models and occasionally in humans. Amongst these very different types of HSV vaccine preparation, the use of a vaccine containing mixed HSV-1 glycoproteins incorporated into immunostimulating complexes (ISCOMs) carrier adjuvant has been shown to provide significant protection in mice and guinea-pigs against spread of virus to the PNS and establishment of latency there [11,12,13,15, 18,33]. The aim of the present study was to determine whether the CNS of mice is protected by immunisation with ISCOMs against infection by HSV-1. 2. Methods 2.1. Animals and vaccination Formulation of HSV-1 ISCOMs, using HSV-1 strain F, was carried out by procedures described previously [11]. The ISCOMs were further purified by density gradient cen-
trifugation. The protein product comprised 97% glycoprotein-types B, C, D, E, H, and I, and an insignificant amount of DNA [12]. BALB/c mice were vaccinated sub-cutaneously with 5 g ISCOMs protein. “Control” animals were injected with phosphate-buffered saline (PBS), pH 7.2 (PBS). Four weeks later, some of the vaccinated animals, and also unvaccinated animals, were infected with a non-lethal dose of HSV-1 (4 ⫻ 105 PFU, strain SC16) by ear scarification. (Strain SC16 was used for injection as it is the most effective in establishing infection in the trigeminal ganglia after ear scarification, while “F” strain was used for vaccine production because it yields a high virus titre; however, antigenically, all HSV-1 strains are identical.) Subsequently, the infected, unvaccinated animals were observed daily over 14 days for ear erythema, and after recovery from the initial injection erythema was scored according to severity on a scale ranging from zero to 4; scores of 3 and 4 were considered significant and indicated that the mouse trigeminal ganglia were infected with HSV-1 [17]. Brains were removed five months post-infection; this time period permits the establishment of viral latency. Great care was taken to prevent any contamination of brains by trigeminal ganglia and any crosscontamination of specimens: separate sterile instruments were used for each tissue and for every animal, and separate rooms used, on successive days, for each group of animals. 2.2. DNA preparation and PCR DNA was prepared as described previously [22] from coded mouse brains (these were minced with a sterile needle and then half was used). PCR assays [22] were carried out “blind.” A 110 bp sequence of the HSV1 thymidine kinase (TK) gene and a 267 bp sequence of the mouse HGPRT gene were amplified in the same tube (the latter was used to check that PCR had proceeded satisfactorily). The 50 l reaction mixture contained 1 g DNA, 2.5 units of Taq DNA polymerase, 200 M of each of the four dNTPs, 2.5 mM Mg2⫹, 500 nM TK primer pairs, 100nM HGPRT primer pairs, and 5l 10 ⫻ reaction buffer (Promega). The mixtures were heated for 5 min at 94°C to denature the DNA and then subjected to 30 cycles of annealing (30 sec at 55°C), extension (30 sec at 72°C) and denaturation (1 min at 94°C), and a final extension step (7 min at 72°C). PCR products were subjected to agarose gel electrophoresis, the DNA being visualised by ethidium bromide. The identity of the viral TK sequence was verified as described previously [23,29]. The sensitivity of detection was approximately one copy of the sequence per 105 cells [23]. Very stringent precautions were taken against contamination during the preparation of DNA and during the PCR procedures, as described previously in detail [22,29]. Standard positive and negative DNA samples for PCR were prepared respectively from HSV1-infected and uninfected Vero cells. Reagent blanks were always included during PCR to check that no cross-contamination had occurred.
W.R. Lin et al. / Neurobiology of Aging 22 (2001) 699 –703
701
Table 1 The relationship between recurrent ear erythema and HSV1 infection in the brain
Recurrent ear erythema No recurrent ear erythema
HSV1 infection in the brain
No HSV1 infection in brain
3 4
3 2
Values indicate number of mice.
ification (Table 1). Statistical analysis using the 2 test, with continuity correction for the small sample size, shows that there was no correlation between extent of erythema and HSV-1 infection in the brain (p ⬎ 0.5). Fig. 1. Electrophoresis of amplified products after PCR for detection of HSV1 TK in brain from ISCOM-vaccinated or unvaccinated mice infected with HSV-1. M is a molecular size marker; lanes 1 and 2, DNA from vaccinated mice; 3– 6, DNA from unvaccinated animals; lane 7, a reagent “blank”; lane 8, no specimen; lane 9, DNA from HSV1-infected Vero cells as a TK “standard.” (Bands below the TK band are primers or aggregates thereof.)
3. Results PCR for the detection of a sequence in the HSV-1 TK gene was carried out on DNA prepared from brains of mice infected with the virus by ear scarification. Fig. 1 shows typical results of agarose gel electrophoresis; in this set, the TK band is present in one unvaccinated sample, but is absent in both vaccinated samples. No viral DNA was detected in vaccinated but unchallenged animals (n ⫽ 9) (not shown), indicating that there was no contamination during the experiment. We detected HSV-1 DNA sequences in brain specimens from 16 of the 39 unvaccinated mice (41%), but in only 3 of the 41 vaccinated mice (7%) (Fig. 2). The difference is statistically significance (2 test, p ⬍ 0.001). The severity of ear erythema was scored in a group of infected, unvaccinated animals after recovery from the scar-
Fig. 2. Protection of mice from HSV-1 latency in brain by vaccination with ISCOMs.
4. Discussion It has been shown [17] that mice infected with HSV-1 suffer from recurrent ear erythema over a period of several months post-infection and then they either recover completely or suffer neurological symptoms. In some of the mice that recover, the erythema recurs at the site of inoculation — a situation that is analogous to herpes labialis in humans. Our present data suggest that there is no correlation between brain infection and erythema i.e. that in mice showing erythema, the virus does not necessarily enter the brain — in contrast to the positive association between HSV1 latency in the TG and erythema. This is particularly important with regard to our previous data since we have shown that apoE-4 is a risk factor for both herpes labialis and AD. As herpes labialis may be analogous to recurrent ear erythema in mice [19], one can deduce that in those people who suffer from herpes labialis and who have an apoE-4 allele, the virus in the TG might not necessarily travel to the brain, and if not, in such cases AD could be caused only by exogenous virus entering the brain. The ISCOMs vaccine is known to protect mice from subsequent HSV-1 infection: 65% of the animals immunised with the vaccine, but none of the non-vaccinated mice, were found to survive after a lethal dose of HSV-1 [15]. The vaccine diminished also the duration of ear erythema in mice [1], and protected against the establishment of latency in the TG, explantation studies showing that 50% of unvaccinated mice harboured reactivatable HSV-1 in the TG compared to only 20% of vaccinated mice [1]. Our present data indicate that the HSV-1-ISCOM vaccine has a protective effect in mice against HSV-1 infection of the CNS also. The extent of protection appears greater in the CNS than in the PNS — 41% of unvaccinated animals harbouring virus compared to only 7% of those vaccinated — but strict comparison can not be made because of the difference in techniques used (PCR and explantation, respectively). The precise pathways used by HSV-1 to infect the brain are uncertain. It might occur via various routes — travelling to
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the brain by retro-axonal transport after reactivation from the TG or other sensory ganglia, or through transport in blood and infection of the brain via the blood-brain barrier (although there is no evidence for that route). However, irrespective of which pathway HSV-1 takes from the ear to the brain, HSV-1 ISCOMs appear to provide protection against establishment of latency in the CNS. Even though direct extrapolation to humans of these results on mice is premature, our findings do appear very promising for the future use of vaccines against HSV-1 in man, especially as the ISCOMs vaccine contains minimal DNA and no proteins of cellular origin. Further, such vaccines might be used also in adults: our earlier results [20] indicate that the virus probably reaches the CNS in older age, and therefore a vaccine might possibly be of value for protection against HSE and AD in the later years of life. This might be even more relevant in future since the age of primary HSV1 infection is now increasing with increasing socio-economic level, at least in developed countries. Protection at least against HSE should be a major consequence of the usage of such a vaccine. Acknowledgments This work was initiated with funding from a Sir Henry Wellcome Innovative Award and continued with a grant from Research into Ageing. We thank Ms. J Graham for assistance.
References [1] Al-Ghamdi A, Jennings R, Bentley H, Potter CW. Latent HSV1 infection in mice immunised with a zwitterionic detergent-extracted HSV-1 antigen preparation. Arch Virol 1989;108:19 –31. [2] Baringer JR, Pisani P. Herpes simplex virus genomes in human nervous system tissue analysed by PCR. Ann Neurol 1994;36:823–29. [3] Bertrand P, Guillaume D, Hellauer K, Dea D, Lindsay J, Kogan S, Gauthier S, Poirier J. Distribution of herpes simplex virus type 1 DNA in selected areas of normal and Alzheimer’s disease brains: a PCR study. Neurodegeneration 1993;2:201– 8. [4] Burke, RL, Goldbeck C, Ng P, Stanberry L, Ott G, Vannest G. The influence of adjuvant on the therapeutic efficacy of a recombinant genital herpes vaccine. J Infect Dis. 1994;170:1110 –19. [5] Byars NE, Fraser-Smith EB, Pecyk RA, Welch M, Nakano G, Burke RL, Hayward AR, Allison AC. Vaccinating guinea pigs with recombinant glycoprotein D of herpes simplex virus in an efficacious adjuvant formulation elicits protection against vaginal infection. Vaccine 1994;12:200 – 09. [6] Cabrera CV, Wohlenber GC, Openshaw H, Rey-Mendez M, Puga A, Notkins AL. Herpes simplex virus DNA sequences in the CNS of latently infected mice. Nature 1980;288:288 –90. [7] Corder EH, Robertson L, Lannfelt L, Bogdanovic N, Eggertsen G, Wilkins J, Hall C. HIV- infected subjects with the E4 allele for APOE have excess dementia, and peripheral neuropathy. Nat Med 1998;4: 1182– 84. [8] Cribbs DH, Azizeh BY, Cotman CW, LaFerla FM. Fibril formation and neurotoxicity by a herpes simplex virus glycoprotein B fragment with homology to the Alzheimer’s A peptide. Biochemistry 2000; 39:5988 –94.
[9] Dobson CB, Itzhaki RF. Herpes simplex virus type 1 and Alzheimer’s disease. Neurobiol. Aging 1999;20:457– 65. [10] Drummond CWE, Eglin RP, Esiri MM. Herpes simplex virus encephalitis in a mouse model: PCR evidence for CNS latency following acute infection. J Neurol Sci. 1994;127:159 – 63. [11] Erturk M, Jennings R, Hockley D, Potter CW. Antibody responses and protection in mice immunised with herpes simplex virus type-1 antigen immune stimulating complex preparations. J Gen Virol. 1989; 21:49 –55. [12] Erturk M, Jennings R, Phillpotts RJ, Potter CW. Biochemical characterization of herpes simplex virus type-1-immunostimulating complexes (ISCOMs): a multi-glycoprotein structure. Vaccine 1991;9: 668 –74. [13] Erturk M, Hill TJ, Shimeld C, Jennings R. Acute and latent infection of mice immunised with HSV-1 ISCOM vaccine. Arch Virol 1992; 125:87– 01. [14] Gallichan WS, Rosenthal KL. Specific secretory immune responses in the female genital tract following intranasal immunisation with a recombinant adenovirus expressing glycoprotein B of herpes simplex virus. Vaccine 1995;13:1589 –95. [15] Ghazi HO, Erturk M, Stannard LM, Faulkner M, Potter CW, Jennings R. Immunogenicity of influenza, and HSV-1 mixed antigen ISCOMs in mice. Arch Virol. 1995;140:1015–31. [16] Gordon L, McQuaid S, Cosby SL. Detection of herpes simplex virus (types 1 and 2) and human herpes virus 6 DNA in human brain tissue by PCR. Clin. Diagn Virol. 1996;6:33– 40. [17] Harbour DA, Hill TJ, Blyth WA. Recurrent herpes simplex in the mouse: inflammation in the skin, and activation of virus in the ganglia following peripheral stimulation. J Gen. Virol. 1983;64:1491–98. [18] Hassan Y, Brewert JM, Alexander J, Jennings R. Immune responses in mice induced by HSV-1 glycoproteins presented with ISCOMs or NISV delivery systems. Vaccine. 1996;14:581– 89. [19] Hill TJ, Field HJ, Blyth WA. Acute and recurrent infection with herpes simplex virus in the mouse: a model for studying latency and recurrent disease. J Gen Virol. 1975;28:341–53. [20] Itzhaki RF, Maitland NJ, Wilcock GK, Yates CM, Jamieson GA. Detection by polymerase chain reaction of herpes simplex virus type 1 (HSV1) DNA in brain of aged normals and Alzheimer’s disease patients. In: Corain B, Iqbal K, Nicolini M, editors. Alzheimer’s Disease: Advances in Clinical, and Basic Research. New York: Wiley & Sons, 1993; p. 97. [21] Itzhaki RF, Lin WR, Shang D, Wilcock GK, Faragher B, Jamieson GA. Herpes simplex virus type 1 in brain, and risk of Alzheimer’s disease. Lancet 1997;349:241– 44. [22] Jamieson GA, Maitland NJ, Wilcock GK, Craske J, Itzhaki RF. Latent herpes simplex virus type 1 in normal, and Alzheimer’s disease brains. J Med Virol. 1991;33:224 –27. [23] Jamieson GA, Maitland NJ, Wilcock GK, Yates, CM, Itzhaki RF. Herpes simplex virus type 1 DNA is present in specific regions of brain from aged people with, and without senile dementia of the Alzheimer type. J Pathol. 1992;167:365– 68. [24] Johnson RT. Viral infection of the nervous systems. Philadelphia: Lippincott-Raven, 1998. [25] Karem KL, Bowen J, Kuklin L, Rouse BT. Protective immunity against herpes simplex (HSV) type 1 following oral administration of recombinant Salmonella typhimurium vaccine strains expressing HSV antigens. J Gen Virol. 1997;78:427–34. [26] Kastrukoff L, Long C, Doherty PC, Wroblewska Z, Koprowski H. Isolation of virus from brain after immunosuppression of mice with latent herpes simplex. Nature 1981;291:432–33. [27] Knotts FB, Cook ML, Stevens JG. Latent herpes simplex virus in the central nervous system of rabbits, and mice. J Exp Med. 1973;138: 740 – 44. [28] Kuklin N, Daheshia M, Karem K, Manickan E, Rouse BT. Induction of mucosal immunity against herpes simplex virus by plasmid DNA immunisation. J Virol. 1997;71:3138 –145.
W.R. Lin et al. / Neurobiology of Aging 22 (2001) 699 –703 [29] Lin WR, Graham J, MacGowan SM, Wilcock GK, Itzhaki RF. Alzheimer’s disease herpes virus in brain, apolipoprotein E4, and herpes labialis. Alzheimer’s Reports. 1998;1:173–78. [30] McLean CS, Erturk M, Jennings R, Nichallanain D, Minson, AC, Duncan I, Bournsell MEG, Inglis SC. Protective vaccination against primary and recurrent disease caused by herpes simplex virus (HSV) type 2 using a genetically disabled HSV-1. J Infect Dis. 1994;170: 1100 – 09. [31] McClements WL, Armstrong, ME, Keys, RD, Liu MA. The prophylactic effect of immunisation with DNA encoding herpes simplex virus glycoproteins on HSV-induced disease in guinea-pigs. Vaccine 1997;15:857– 60. [32] Morrison LA, Knipe DM. Immunisation with replication-defective mutants of herpes simplex virus type 1: sites of immune intervention in pathogenesis of challenge virus infection. J Virol. 1994;68:689– 96.
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[33] Simms JR, Heath AW, Jennings R. Use of herpes simplex virus (HSV) type 1 ISCOMS vaccine for prophylactic, and therapeutic treatment. J Infect Dis 2000;181:1240 – 48. [34] Stevens JG, Cook ML. Latent herpes simplex virus in spinal ganglia of mice. Science. 1971;173:843– 45. [35] Walker J, Leib DA. Protection from primary infection and establishment of latency by vaccination with a herpes simplex virus type 1 recombinant deficient in the virion host shutoff (vhs) function. Vaccine 1998;16:1–5. [36] Walz M, Price RR, Notkins A. Latent ganglionic infection with herpes simplex virus types 1, and 2: viral reactivation in virus after neurectomy. Science 1974;184:1185– 87. [37] Zheng BJ, Graham FL, Johnson DC, Hanke T, McDermott MR, Prevec L. Immunogenicity in mice of tandem repeats of an epitope from herpes simplex gD protein when expressed by recombinant adenovirus vectors. Vaccine 1993;11:1191–98.
www.elsevier.com/locate/neuaging
Open peer commentary
Vaccination prevents latent HSV1 infection of mouse brain Woan-Ru Lina,1, Roy Jenningsb, Trixie L. Smithb, Matthew A. Wozniaka, Ruth F. Itzhakia,* a
University of Manchester Institute of Science & Technology, Molecular Neurobiology Laboratory, Department of Optometry & Neuroscience, Manchester M60 1QD, UK b University of Sheffield Medical School, Division of Molecular & Genetic Medicine, Sheffield S10 2RX, UK Received 26 July 2000; received in revised form 20 October 2000; accepted 22 December 2000
Abstract Herpes simplex encephalitis (HSE) is a rare but very serious disorder caused by herpes simplex type 1 virus (HSV-1). Treatment with acyclovir decreases mortality but many patients still suffer cognitive impairment subsequently. A vaccine against HSV1 would therefore be of great value. HSV-1 has been implicated also in Alzheimer’s disease (AD): we established that HSV1 resides in the brain of about two thirds of AD patients and aged normal people, and that in carriers of the type 4 allele of the apolipoprotein E gene, it is a strong risk factor for AD. Thus a vaccine against HSV-1 might prevent development of AD in some cases. To find whether a vaccine of mixed HSV-1 glycoproteins (ISCOMs), which protects mice from latent HSV-1 infection of sensory ganglia, prevents HSV1 latency in the CNS, ISCOM-vaccinated or unvaccinated animals were infected with HSV-1. Using polymerase chain reaction (PCR) we detected HSV-1 in brain from 16 of 39 unvaccinated mice (41%), but only 3 of 41 vaccinated mice (7%) (P ⬍ 0.001). Thus, ISCOMs protect the CNS also, suggesting their possible future usage in humans. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Herpes simplex type 1 virus; Vaccine; Mice; Brain; Herpes simplex encephalitis; Alzheimer’s disease; PCR
1. Introduction Primary herpes simplex type 1 virus (HSV-1) infection usually occurs during childhood, with infection typically developing as a result of the direct inoculation of infected droplets from orolabial or nasal secretions on to susceptible mucosal surfaces. HSV-1 infection is virtually universal — most adults are seropositive. Following initial infection with HSV-1, the virus establishes latent infection in the trigeminal ganglia (TG), from which the virus reactivates periodically causing recurrent disease, manifested as cold sores (herpes labialis), in certain people [24]. However, there are many individuals with no history of any disorder caused by HSV-1 infection yet who show evidence of prior infection. HSV-1 is responsible also for most cases of herpes simplex encephalitis (HSE) [24], a brain disease which has an annual incidence estimated at between 1 in 250000 and 1 in a million of the population. HSE causes high mortality as well * Corresponding author. Tel.: ⫹00-44-161-200-3879; fax: ⫹00-44161-200-4433. E-mail address: [email protected] (R.F. Itzhaki). 1 Current address: Epilepsy Unit and Neuropathology Laboratory, Institute of Neurology, Queen’s Square, London WC1N 3BG.
as significant morbidity in survivors. Even though treatment of HSE with acyclovir in the early stages of the disease decreases mortality, patients are not free of further complications. Many suffer from severe cognitive impairments which can seriously affect their quality of life. Therefore it is of great importance to develop a vaccine against HSV-1 so that the suffering caused by both herpes labialis and the more serious condition, HSE, is alleviated or even prevented. Our previous studies established that HSV-1 can reside latently within the brains of a high proportion of aged people [22,23]; it has a predilection for the temporal lobe, frontal lobe and hippocampus. Subsequent studies by other groups have confirmed the presence of HSV1 in human brain [2,3,16]. We have since found that the combination of HSV-1 in brain and carriage of the type 4 allele of the apolipoprotein E gene (apoE-4) is a strong risk factor for Alzheimer’s disease (AD), and that neither HSV-1 nor apoE-4 alone is a risk [21,29]. We found also, in striking parallelism in the peripheral nervous system (PNS), that apoE-4 is a risk factor for herpes labialis [21,29]. Another link between HSV1 and AD is the recent finding that a fragment of HSV1 glycoprotein B has sequence homology to the A pepide found in the characteristic plaques of AD brain [8]. As to interactions between viruses and lipopro-
0197-4580/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 1 9 7 - 4 5 8 0 ( 0 1 ) 0 0 2 3 9 - 1
700
W.R. Lin et al. / Neurobiology of Aging 22 (2001) 699 –703
teins (or their components), other cases have been described in diverse types of virus (see review, ref. [9]), the most relevant being between apoE-4 and the onset of dementia and peripheral neuropathy in HIV-infected (pre-AIDS) subjects [7]. On the basis of our findings in AD, we suggested that the combination of virus and apoE-4 is particularly harmful in the nervous system and that the virus in brain reactivates periodically, causing more damage — and eventually AD — in apoE-4 carriers. HSV-1 in brain is the first environmental agent to be definitely implicated in AD, and our results strengthen the rationale for developing a vaccine against HSV-1. As to HSV-1 infection of the mouse nervous system, the discovery that administration of HSV-1 at various sites caused acute infection and later, establishment of a latent infection in the PNS, led to usage of mice as a model for reactivation of the virus in the PNS of humans [19,34,36]. Studies on the mouse central nervous system (CNS), using explantation, co-cultivation, or molecular hybridisation, showed that after HSV-1 infection, viral latency could be established in brain also, following acute infection [6,26, 27]; it was therefore suggested that this might be a suitable model for investigating neurological diseases with a known or a possible viral aetiology. More recently, HSV1-infected mice have been used as a model for HSE, seeking the viral DNA and viral transcripts in brain by PCR [10]. Vaccines against HSV-1 have taken several forms over the years. Initially, inactivated vaccines derived from phenol-treated tissues from infected animals were used, but these contained animal proteins and their administration led to demyelination; hence this procedure was not pursued further. Subsequently, ultraviolet light-inactivated purified viruses derived from tissue culture were used. However, the vaccine was not effective and sometimes caused side effects. In more recent years, replication-defective HSV-1 vaccines [30,32,35], vectored HSV glycoproteins-usually gD or gB [14,25,37], DNA vaccines [28,31] and adjuvanted glycoprotein vaccine preparations [4,5] have been assessed in animal models and occasionally in humans. Amongst these very different types of HSV vaccine preparation, the use of a vaccine containing mixed HSV-1 glycoproteins incorporated into immunostimulating complexes (ISCOMs) carrier adjuvant has been shown to provide significant protection in mice and guinea-pigs against spread of virus to the PNS and establishment of latency there [11,12,13,15, 18,33]. The aim of the present study was to determine whether the CNS of mice is protected by immunisation with ISCOMs against infection by HSV-1. 2. Methods 2.1. Animals and vaccination Formulation of HSV-1 ISCOMs, using HSV-1 strain F, was carried out by procedures described previously [11]. The ISCOMs were further purified by density gradient cen-
trifugation. The protein product comprised 97% glycoprotein-types B, C, D, E, H, and I, and an insignificant amount of DNA [12]. BALB/c mice were vaccinated sub-cutaneously with 5 g ISCOMs protein. “Control” animals were injected with phosphate-buffered saline (PBS), pH 7.2 (PBS). Four weeks later, some of the vaccinated animals, and also unvaccinated animals, were infected with a non-lethal dose of HSV-1 (4 ⫻ 105 PFU, strain SC16) by ear scarification. (Strain SC16 was used for injection as it is the most effective in establishing infection in the trigeminal ganglia after ear scarification, while “F” strain was used for vaccine production because it yields a high virus titre; however, antigenically, all HSV-1 strains are identical.) Subsequently, the infected, unvaccinated animals were observed daily over 14 days for ear erythema, and after recovery from the initial injection erythema was scored according to severity on a scale ranging from zero to 4; scores of 3 and 4 were considered significant and indicated that the mouse trigeminal ganglia were infected with HSV-1 [17]. Brains were removed five months post-infection; this time period permits the establishment of viral latency. Great care was taken to prevent any contamination of brains by trigeminal ganglia and any crosscontamination of specimens: separate sterile instruments were used for each tissue and for every animal, and separate rooms used, on successive days, for each group of animals. 2.2. DNA preparation and PCR DNA was prepared as described previously [22] from coded mouse brains (these were minced with a sterile needle and then half was used). PCR assays [22] were carried out “blind.” A 110 bp sequence of the HSV1 thymidine kinase (TK) gene and a 267 bp sequence of the mouse HGPRT gene were amplified in the same tube (the latter was used to check that PCR had proceeded satisfactorily). The 50 l reaction mixture contained 1 g DNA, 2.5 units of Taq DNA polymerase, 200 M of each of the four dNTPs, 2.5 mM Mg2⫹, 500 nM TK primer pairs, 100nM HGPRT primer pairs, and 5l 10 ⫻ reaction buffer (Promega). The mixtures were heated for 5 min at 94°C to denature the DNA and then subjected to 30 cycles of annealing (30 sec at 55°C), extension (30 sec at 72°C) and denaturation (1 min at 94°C), and a final extension step (7 min at 72°C). PCR products were subjected to agarose gel electrophoresis, the DNA being visualised by ethidium bromide. The identity of the viral TK sequence was verified as described previously [23,29]. The sensitivity of detection was approximately one copy of the sequence per 105 cells [23]. Very stringent precautions were taken against contamination during the preparation of DNA and during the PCR procedures, as described previously in detail [22,29]. Standard positive and negative DNA samples for PCR were prepared respectively from HSV1-infected and uninfected Vero cells. Reagent blanks were always included during PCR to check that no cross-contamination had occurred.
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Table 1 The relationship between recurrent ear erythema and HSV1 infection in the brain
Recurrent ear erythema No recurrent ear erythema
HSV1 infection in the brain
No HSV1 infection in brain
3 4
3 2
Values indicate number of mice.
ification (Table 1). Statistical analysis using the 2 test, with continuity correction for the small sample size, shows that there was no correlation between extent of erythema and HSV-1 infection in the brain (p ⬎ 0.5). Fig. 1. Electrophoresis of amplified products after PCR for detection of HSV1 TK in brain from ISCOM-vaccinated or unvaccinated mice infected with HSV-1. M is a molecular size marker; lanes 1 and 2, DNA from vaccinated mice; 3– 6, DNA from unvaccinated animals; lane 7, a reagent “blank”; lane 8, no specimen; lane 9, DNA from HSV1-infected Vero cells as a TK “standard.” (Bands below the TK band are primers or aggregates thereof.)
3. Results PCR for the detection of a sequence in the HSV-1 TK gene was carried out on DNA prepared from brains of mice infected with the virus by ear scarification. Fig. 1 shows typical results of agarose gel electrophoresis; in this set, the TK band is present in one unvaccinated sample, but is absent in both vaccinated samples. No viral DNA was detected in vaccinated but unchallenged animals (n ⫽ 9) (not shown), indicating that there was no contamination during the experiment. We detected HSV-1 DNA sequences in brain specimens from 16 of the 39 unvaccinated mice (41%), but in only 3 of the 41 vaccinated mice (7%) (Fig. 2). The difference is statistically significance (2 test, p ⬍ 0.001). The severity of ear erythema was scored in a group of infected, unvaccinated animals after recovery from the scar-
Fig. 2. Protection of mice from HSV-1 latency in brain by vaccination with ISCOMs.
4. Discussion It has been shown [17] that mice infected with HSV-1 suffer from recurrent ear erythema over a period of several months post-infection and then they either recover completely or suffer neurological symptoms. In some of the mice that recover, the erythema recurs at the site of inoculation — a situation that is analogous to herpes labialis in humans. Our present data suggest that there is no correlation between brain infection and erythema i.e. that in mice showing erythema, the virus does not necessarily enter the brain — in contrast to the positive association between HSV1 latency in the TG and erythema. This is particularly important with regard to our previous data since we have shown that apoE-4 is a risk factor for both herpes labialis and AD. As herpes labialis may be analogous to recurrent ear erythema in mice [19], one can deduce that in those people who suffer from herpes labialis and who have an apoE-4 allele, the virus in the TG might not necessarily travel to the brain, and if not, in such cases AD could be caused only by exogenous virus entering the brain. The ISCOMs vaccine is known to protect mice from subsequent HSV-1 infection: 65% of the animals immunised with the vaccine, but none of the non-vaccinated mice, were found to survive after a lethal dose of HSV-1 [15]. The vaccine diminished also the duration of ear erythema in mice [1], and protected against the establishment of latency in the TG, explantation studies showing that 50% of unvaccinated mice harboured reactivatable HSV-1 in the TG compared to only 20% of vaccinated mice [1]. Our present data indicate that the HSV-1-ISCOM vaccine has a protective effect in mice against HSV-1 infection of the CNS also. The extent of protection appears greater in the CNS than in the PNS — 41% of unvaccinated animals harbouring virus compared to only 7% of those vaccinated — but strict comparison can not be made because of the difference in techniques used (PCR and explantation, respectively). The precise pathways used by HSV-1 to infect the brain are uncertain. It might occur via various routes — travelling to
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the brain by retro-axonal transport after reactivation from the TG or other sensory ganglia, or through transport in blood and infection of the brain via the blood-brain barrier (although there is no evidence for that route). However, irrespective of which pathway HSV-1 takes from the ear to the brain, HSV-1 ISCOMs appear to provide protection against establishment of latency in the CNS. Even though direct extrapolation to humans of these results on mice is premature, our findings do appear very promising for the future use of vaccines against HSV-1 in man, especially as the ISCOMs vaccine contains minimal DNA and no proteins of cellular origin. Further, such vaccines might be used also in adults: our earlier results [20] indicate that the virus probably reaches the CNS in older age, and therefore a vaccine might possibly be of value for protection against HSE and AD in the later years of life. This might be even more relevant in future since the age of primary HSV1 infection is now increasing with increasing socio-economic level, at least in developed countries. Protection at least against HSE should be a major consequence of the usage of such a vaccine. Acknowledgments This work was initiated with funding from a Sir Henry Wellcome Innovative Award and continued with a grant from Research into Ageing. We thank Ms. J Graham for assistance.
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