JURNAL BELL’S PALSY
Abstract
Background
An association was
previously established between facial nerve paralysis (Bell's palsy) and
intranasal administration of an inactivated influenza virosome vaccine
containing an enzymatically active Escherichia coli Heat
Labile Toxin (LT) adjuvant. The individual component(s) responsible for
paralysis were not identified, and the vaccine was withdrawn.
Methodology/Principal
Findings
Subjects participating
in two contemporaneous non-randomized Phase 1 clinical trials of nasal subunit
vaccines against Human Immunodeficiency Virus and tuberculosis, both of which
employed an enzymatically inactive non-toxic mutant LT adjuvant (LTK63),
underwent active follow-up for adverse events using diary-cards and clinical
examination. Two healthy subjects experienced transient peripheral facial nerve
palsies 44 and 60 days after passive nasal instillation of LTK63, possibly a
result of retrograde axonal transport after neuronal ganglioside binding or an
inflammatory immune response, but without exaggerated immune responses to
LTK63.
Conclusions/Significance
While the unique anatomical predisposition of the facial nerve
to compression suggests nasal delivery of neuronal-binding LT–derived adjuvants
is inadvisable, their continued investigation as topical or mucosal adjuvants
and antigens appears warranted on the basis of longstanding safety via oral,
percutaneous, and other mucosal routes.
Citation: Lewis DJM, Huo Z, Barnett S, Kromann I, Giemza
R, et al. (2009) Transient Facial Nerve Paralysis (Bell's Palsy) following
Intranasal Delivery of a Genetically Detoxified Mutant ofEscherichia coli Heat
Labile Toxin. PLoS ONE 4(9): e6999. doi:10.1371/journal.pone.0006999
Editor: Stefan Bereswill, Charité-Universitätsmedizin
Berlin, Germany
Received: June 16, 2009; Accepted: July
29, 2009; Published: September 16, 2009
Copyright: © 2009 Lewis et al. This is an
open-access article distributed under the terms of the Creative Commons
Attribution License, which permits unrestricted use, distribution, and
reproduction in any medium, provided the original author and source are
credited.
Funding: This work was funded by a grant from the
Commission of the European Union Sixth Framework Programme, “MUVAPRED”:
FP6-2002-LIFESCIHEA-2.3 503240,http://cordis.europa.eu/fp7. The work at
Novartis was supported by an HIV Vaccine Design and Development Teams contract
NO-1-AI-05396 from the Division of AIDS-NIAID-NIH,http://www3.niaid.nih.gov/about/organization/daids/.
The funders had input on study design, but had no role in data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing interests: David J.M. Lewis, Zhiming Huo, Rafaela
Giemza, Maria Woodrow and Eva Galiza are employees of St George's - University
of London which co-sponsored trial 2005-005140-81. Susan Barnett, Deborah
Novicki, Giuseppe Del Giudice and Rino Rappuoli are employees of Novartis
Vaccines which has intellectual property rights on LTK63, and co-sponsored
trial 2005-005140-81. Ingrid Kromann, Birgit Thierry-Carstensen and Peter
Andersen are employees of Staten Serum Institute which has intellectual
property rights on Ag85-ESAT6, and sponsored trial 2005-005983-10.
Mucosal immunization
via intranasal route has received much attention in recent years [1] due to potential advantages of
needle-free delivery and enhanced mucosal immune responses against infections
such as influenza or Human Immunodeficiency Virus (HIV), which can be enhanced
by co-administration of effective mucosal adjuvants such as mutants of Escherichia
coli heat labile toxin (LT) [2]. However, this immunization approach
raised concerns after a strong epidemiological association was reported [3] between facial nerve paralysis
(Bell's Palsy) and intranasal administration of inactivated influenza virosome
vaccine “NasalFlu”containing an enzymatically active mutant LT
adjuvant [4]. Retrospective analysis did not identify
relative contributions of vaccine components [3], and NasalFlu was
withdrawn. We report two cases of transient Bell's Palsy following nasal
administration of a genetically detoxified LT adjuvant (LTK63) [2] during two Phase 1 vaccine trials.
Objectives and
participants
The cases reported here occurred during two contemporaneous,
open-label, Phase 1 clinical vaccine trials of safety and immunogenicity at the
same clinical site (St George's Vaccine Institute, London). No formal sample
size calculations were made, as is usual in this early stage of evaluation.
Full details of trials outcomes are in preparation for publication. Briefly,
nine BCG vaccine-naive healthy subjects aged 18–45 were entered into a
non-randomized Phase 1 trial of a nasal Mycobacterium tuberculosis subunit
vaccine [5] (full protocol available on
ClinicalTrials.gov NCT00440544: http://clinicaltrials.gov/ct2/show/NCT00440544,
EudraCT: 2005-005983-10). The first four subjects received two nasal
immunizations (days 0 and 56) with 100 µg Mycobacterium tuberculosis Ag85B-ESAT6
fusion protein alone, and the subsequent five subjects received two nasal
immunizations (days 0 and 56) of 100 µg Mycobacterium tuberculosis Ag85B-ESAT6
fusion protein mixed with 30 µg LTK63. After the event reported here the trial
was terminated prior to recruitment of subsequent planned groups.
In the contemporaneous trial, 30 healthy subjects aged 18–45
were entered into a non-randomized Phase 1 clinical trial of a nasal HIV
glycoprotein-140 subunit vaccine [6] (full protocol available on
ClinicalTrials.gov NCT00369031: http://clinicaltrials.gov/ct2/show/NCT00369031,
EudraCT: 2005-005140-81), and allocated to one of three consecutively-recruited
groups (10 per group) to receive three intranasal immunisations (days 0, 28,
and 56) with either: 100 µg HIV glycoprotein-140 mixed with 30 µg LTK63, 100 µg
HIV glycoprotein-140 alone, or 30 µg LTK63 alone. All subjects received two
intramuscular immunizations with 100 µg HIV glycoprotein-140 mixed with MF59
adjuvant [7] on days 112 and 196. Following the
case reported here 4 subjects in the group receiving 30 µg LTK63 alone nasally
underwent only one nasal immunization, but did receive two intramuscular
immunizations. The subject reported here had no further immunizations after the
first nasal immunization with 30 µg LTK63 alone.
Collection,
processing, and analysis of serum and nasal lavage samples for immune responses
to LTK63
Blood and nasal samples were obtained at pre-defined time points
before and after immunization. Blood was collected by antecubital fossa
venepuncture, serum separated and frozen at −20°C. Nasal lavage with 5 mL
sterile water was performed via urinary catheter placed the anterior nares
bilaterally, and samples frozen at −80°C. IgG and IgA antibodies to LTK63 were
detected by indirect ELISA [8], expressed in µg/mL using a human
immunoglobulin standard (NIBSC, UK).
Investigations for
alternative causes of facial nerve paralysis
Pre- and post-immunization serum or plasma samples were
retrospectively analysed for evidence of the most common conditions associated
with Bell's palsy including serology/PCR/antigen detection of infection with
Human Immunodeficiency Virus, Epstein-Barr Virus (EBV), Varicella zoster virus
(VZV), Borrelia burgdorferi, measles, mumps, Herpes simplex 1 and 2
(HSV) and cytomegalovirus (CMV); or autoimmune diseases including antinuclear
antibodies (ANA), anti-neutrophil cytoplasmic antibodies (ANCA) and Angiotensin
Converting Enzyme (ACE) levels. Both subjects declined radiological
examinations.
Ethics
All
subjects provided written informed consent before undergoing screening or
participation in the trials, which were both approved by St George's Research
Ethics Committee and UK Medicines and Healthcare products Regulatory Agency. In
addition the two subjects reported here provided additional written consent for
publication of details which may include identifying information.
Case 1
A healthy 29 year old, white male enrolled into nasal Mycobacterium
tuberculosis subunit vaccine trial 2005-005983-10. He reported an
uncomplicated nasal procedure age 6, insertion of tympanic membrane grommets in
childhood, mild conjunctivitis when pollen count high, mild itching after
prolonged grass contact, but no seasonal rhinitis, neurological disease,
migraine or regular medications. Screening HIV, hepatitis B and C serology,
hematology and biochemistry were negative or normal. Mantoux test and
‘QuantiFERON-TB Gold’ (Cellestis, UK) were negative for latent tuberculosis.
Medical examination including ear, nose and throat (ENT) and cranial nerves was
normal apart from scar on right tympanic membrane. Chest radiograph was normal.
He received 100 µg Mycobacterium tuberculosis Ag85B-ESAT6
fusion protein and 30 µg LTK63, in 300 µL physiological buffer, by passive
instillation of drops divided equally between right and left anterior nares. A
diary was issued to record symptoms.
On day 1 he recorded mild malaise, ENT discomfort, and cough.
Examination on day 7 was normal. Between days 34–43 he recorded mild itching of
eyes and took loratidine. Examination on day 43 was normal. Between days 45–48
he experienced mild coryza and nasal discharge treated with acetaminophen.
Examination on day 56 was normal, and he received second nasal immunization
with Ag85B-ESAT6 and LTK63 as before. Between days 57–61 took loratidine for a
sensation of fluid in right ear. On day 58 he experienced a migraine-like
visual aura bilaterally, lasting 20 minutes. On day 60 he noticed mild right
sided post-auricular pain, followed by abnormality of taste, worsening over day
61. On day 62 (5 days after second immunization) he developed right-sided
facial asymmetry and increased tear production.
On examination on day 64 he was afebrile, cardiovascular,
respiratory, abdominal examination normal, no mastoid tenderness, rashes or
peri-auricular vesicles. ENT normal except clear fluid behind non-inflamed
right tympanic membrane. Sense of smell normal on testing. Cranial nerves 2–6
normal, corneal reflex intact, pupils equal and responsive, fundoscopy normal.
House-Brackmann [9] grade V signs of right lower motor
neurone facial nerve palsy present, with inability to taste salt, sour, and
bitter on right anterior portion of tongue, and profuse tear production (seen
in 67% of Bell's palsies [10] as orbicularis oculis dysfunction
prevents tear transport to lacrimal sac, without hypersecretion). Marked Bell's
phenomenon (upward gaze upon attempting to close affected eye) observed,
confirming peripheral lesion [11]. Hearing and balance normal, Rinne's
normal, Weber localized to right (seen in Bell's palsy, also compatible with
observed middle ear fluid). Cranial nerves 9–12 normal. Sensation over head and
neck normal. He was prescribed prednisolone, omeprazole, aciclovir (8 days),
eye drops & patches according to UK guidance [12]. Palsy was graded III on day 70, and II
by day 78 without increased tearing, and steroids discontinued. Symptoms of
right retro-auricular pain and abnormality of taste recurred between days 87–89
(30–32 after second immunization) without facial paralysis. He recommenced
prednisolone and aciclovir for 11 and 7 days respectively. On day 91
examination was normal except slight abnormality of taste. Examination on days
126 and 225 (168 days after second immunization) were normal.
There were no clinically significant abnormalities of hematology
and biochemistry throughout study. Screening and day 225 samples were tested
for: HIV 1&2 antibody, p24 antigen (negative); VZV (IgM negative, IgG
positive, no rise in titer); B. burgdorferi (IgG and IgM
negative); measles, mumps (IgG positive, no rise in titer); HSV 1 and 2 (IgG
negative); CMV (IgG and IgM negative); ANA, ANCA (negative). ACE 22 U/L day 56,
and 16 day 91 (normal range 8–52). Anti-BV Nuclear Antigen positive on
screening and on day 225, no rise in titer; anti-EBV Early Antigen IgG 52 at
screening rising to 150 on day 225; anti-EBV Early Antigen IgM 23·5 (weak
positive) at screening and 14·2 (negative) on day 225. This pattern suggests
possibility of EBV infection in months before screening.
Case 2
A healthy 20 year old, white female enrolled into nasal HIV
glycoprotein-140 subunit vaccine trial 2005-005140-81. She gave no history of
neurological or otolaryngology disease, was taking no regular medications
except oral contraceptives. Screening HIV, hepatitis B and C serology, plasma
HIV nucleic acid, hematology and biochemistry panels, blood and urine pregnancy
tests were negative or normal. Physical examination was normal including ENT
and cranial nerves. She received 30 µg of LTK63 (as a nasal placebo) in 284 µL
physiological buffer, by passive instillation of drops divided equally between
right and left anterior nares. A symptoms diary was issued.
On days 2 and 3 she recorded mild malaise, nasal congestion and
headache, resolved with ibuprofen. Examination on day 3 was normal. Between
days 8–11 she reported mild headache, resolved by stat acetaminophen and
pseudoephedrine. On day 28 she was afebrile, physical examination normal;
hematology and biochemistry normal. Her scheduled second (and subsequently third)
immunization was cancelled due to Bell's palsy affecting Case 1. On days 37–40
she reported mild nasal congestion and discharge, on day 43 mild cough, and on
days 44–45 left sided shoulder/neck pain “like a pulled muscle”, and took
diclofenac. On day 44 she developed left retro-auricular pain, stinging of left
eye with increased tearing, and jaw paresthesia, followed by facial asymmetry
and difficulty closing left eye. A local Emergency Room diagnosed Bell's palsy,
but gave no treatment. On day 45 abnormal taste sensation developed and she
attended the trial site. There was no history of trauma, headache, fevers,
aura/visual disturbance, or abnormality of smell. She was afebrile;
cardiovascular, respiratory, abdominal, ENT examinations normal; no mastoid
tenderness, rashes or peri-auricular vesicles. Normal sense of smell on
testing; cranial nerves 2–6 intact; pupils equal and responsive, fundoscopy
normal. A grade IV left lower motor neurone facial nerve palsy was present with
difficulty distinguishing salt, sour and bitter taste on anterior portion of
tongue, and increased tearing. Hearing and balance, Rinne's and Weber tests
normal. Gag reflex present but decreased on left. Cranial nerves 11–12 normal,
with appearance of tongue deviating to right (feature of Bell's palsy due to
mouth asymmetry, not twelfth nerve defect). Sensation over head and neck
normal. She was prescribed prednisolone, omeprazole, aciclovir, eye drops &
patches [12]. Grade II weakness was present on day 44,
with reduced taste sensation. A Consultant Neurologist confirmed the diagnosis
on day 48. Palsy almost completely resolved by day 50, and steroids were
discontinued, with complete recovery by day 58. She was discharged on day 214
with no further relapse.
There were no clinically significant abnormalities of hematology
and biochemistry throughout study. Screening and day 224 samples were tested
for: HIV 1&2 antibody, p24 antigen and HIV nucleic acid PCR (negative); VZV
(IgG positive, no rise in titer, IgM negative); B. burgdorferi(IgG
and IgM negative); measles (IgG positive, no rise in titer); mumps, HSV 1 and 2
(IgG negative); EBV (EBNA IgG positive, no rise in titer, EBV Early Antigen IgG
and IgM negative); CMV (IgG and IgM negative); ANA, ANCA (negative). ACE 42 U/L
day 27, 24 day 55 (normal range 8–52).
Immune responses to
LTK63
LT is a potent mucosal antigen [13] therefore antibody responses to
LTK63 were measured using an in-house ELISA on serum and nasal lavage samples
obtained from subjects before and after immunization. IgG and IgA responses to
LTK63 were observed after each immunization (Figure 1), and although numbers in each group
are small, responses in cases experiencing Bell's palsy were generally
comparable with group mean.
Discussion
We report two cases of Bell's palsy temporally associated with
nasal administration of non-toxic LTK63, possibly a result of transient
interference with peripheral nerve function due to accumulation of LTK63
molecules, or inflammation arising from immune response to LTK63, following
ganglioside binding and retrograde neuronal transport; or another unknown
mechanism. Both subjects rapidly recovered without long-term sequelae. The
situation of our subjects prospectively recording the progressive symptoms and
signs of Bell's palsy from a clearly defined time-point offered an unique
opportunity to study in some detail the pathophysiology of the neurological
disturbance, in contrast with most clinical studies in which symptoms are
recalled retrospectively, and in which there is no clearly defined starting point.
Bell's palsy arises from facial nerve compression within the
facial canal, or acute demyelination, usually associated with herpes infection,
but also other conditions including parenteral immunization, infections, tumors
and autoimmune diseases [11]. Cholera toxin (CT) undergoes retrograde
axonal transport by binding neuronal gangliosides [14], a property shared by LT, but unlike
tetanus toxin CT cannot cross synapses [14]. Considerable attention has focused on
safety issues associated with possible olfactory nerve transport of CT/LT
molecules to the CNS [15], a single cell-system with no synapse
outside the CNS. Whereas human olfactory nerve endings are poorly accessible to
passive instillation of drops into the anterior nares, facial nerve
secretomotor fibers richly supply nasal mucosa, and can transport molecules to
the geniculate ganglion via the nerve of the pterygoid canal and greater
petrosal nerve. The sequential appearance of classical symptoms and signs of
Bell's palsy, without olfactory disturbance we observed, are consistent with
progressive interference of peripheral (not central nervous system) facial
nerve function by a process initiated during retrograde axonal transport.
Intriguingly, secretomotor symptoms preceded both cases, which is of interest
as antecedent symptoms of “upper respiratory tract infection” are reported in
20% of Bell's palsies [16], and often ascribed as a cause rather
than a consequence of the neurological defect. The involvement of trigeminal
and glossopharyngeal nerves in Case 2 is well recognised [16] possibly due to spread of the causal
agent (usually a herpes virus) via superficial greater petrosal nerve [17], which connects facial, trigeminal and
glossopharyngeal nerves. The migraine-like aura without headache in Case 1 is
interesting as carotid plexus autonomic innervation joins the greater petrosal
nerve, and release of vasoactive neuropeptides from trigeminal and
parasympathetic perivascular fibers has been associated with migraine[18]. The onset of symptoms on day 44 and 60
(with a possible relapse 31 days after second immunization in Case 1) is
consistent with the highest risk of palsy between 31 to 60 days after NasalFlu [3], suggesting a common pathogenesis.
An annual Bell's palsy incidence of 32 per 100,000 was reported
in a European population [10], We observed two cases of Bell's palsy in
25 subjects, receiving 59 LTK63 30 µg doses, making a chance association highly
unlikely. Only 13 cases per 10,000 were estimated within 91 days after
vaccination with NasalFlu [3], and no toxicity was seen during 42 day follow-up
in a Phase I trial of a Chiron (now Novartis) intranasal inactivated influenza
vaccine [19] in which 18 subjects received two
doses of 30 µg LTK63 alone; a further 16 two doses of 30 µg LTK63 with
influenza antigens and particulate biovector; and another 30 receiving two
doses of 3 or 10 µg LTK63 with antigens and biovector. It is possible that our
free suspension of LTK63 allowed more molecules to bind neuronal gangliosides
than when complexed with virosomes (NasalFlu) or biovectors
(Chiron-Novartis vaccine), and the absence of symptoms in 18 subjects receiving
LTK63 alone in the Chiron nasal vaccine trial is not significant (Fisher's exact
test: p = 0.52; Odds Ratio 3.2 (0.15–68).
It was
speculated [20] that reactivation of herpes
infection may have been induced by toxicity of the LT used in NasalFlu (which
induced diarrhea when given orally [4]) as extensive preclinical evaluation,
including primates, was unremarkable [21]. We followed standard UK guidance and
both subjects received antiviral and steroid therapy, and rapidly made a full
recovery, although it is not possible to determine whether the treatment
affected the course of the disease. Both cases were seronegative for HSV
(although Case 1 had some weak serological evidence of asymptomatic EBV
infection in the months prior to recruitment), and LTK63 is rendered completely
non-toxic by an amino acid substitution in the enzyme active site that
abolishes ADP-ribosylation [2]. Our vaccines were produced under GMP
conditions, free of contaminants including lipopolysaccharide, and nasal
testing in rabbits, including detailed CNS histology, revealed no localized or
generalized toxicity. Extensive preclinical experience with nasal LTK63[2] has demonstrated mucosal
adjuvanticity without toxicity, including macaques [6], Guinea Pigs [22], and mice [5].
In conclusion, the
unique anatomical predisposition of the facial nerve to compression within the
facial canal suggests that nasal administration of neuronal-binding LT-derived
molecules is inadvisable. However, the extensive clinical safety record of
oral [13] and percutaneous [23]administration of CT/LT molecules provides
a rationale for their continued investigation as potent mucosal and topical
vaccine antigens and adjuvants. The lack of reported facial nerve paralysis
following nasal immunization in subjects not receiving LT adjuvants supports
the continued exploration of nasal delivery as an effective and needle-free
route of immunization against mucosally-acquired infections as influenza and
HIV.
Conceived and designed the experiments: DJML
SB IK BTC PA DN GDG RR. Performed the experiments: DJML ZH RG EG MW. Analyzed
the data: DJML. Contributed reagents/materials/analysis tools: RR. Wrote the
paper: DJML ZH SB IK RG EG MW BTC PA DN GDG RR.
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