Immunokinetics of equine herpesvirus 1 in donkey mares: suppression of secondary

Rev. sci. tech. Off. int. Epiz., 1992, 11 (3), 901-908
Immunokinetics of equine herpesvirus 1
in donkey mares: suppression of secondary
cell-mediated response
M. SINGH and S. C H A R A N *
Summary: To study the immunokinetics of equine herpesvirus 1 (EHVI), donkey
mares were immunised with a laboratory strain of EHV1, or with recommended
doses of Pneumabort-K vaccine (EHV1 Army 183 strain, formalin inactivated,
with an oil adjuvant) and a booster was given after three months.
Humoral immune responses were studied by employing a virus neutralisation
(VN) test. A leucocyte migration inhibition test (LMIT) was employed for the
assay of cellular immune responses.
The VN antibody litre reached 1:64 or 1:128 after primary immunisation
and showed a marginal increase (1:256) after secondary immunisation with either
of the immunogens.
After the primary dose of immunogen, there was a gradual increase in host
cellular response which persisted for up to three months. However, on secondary
immunisation, cell-mediated immune response was short-lived and weak
compared to the primary response with both immunogens. This could be one
possible explanation for breakdown of anti-EHVl immunity leading to abortion
in immunised mares.
KEYWORDS: Equine herpesvirus - Immune response - Immunosuppression
Leucocyte migration inhibition test - Primary and secondary immune
responses Virus neutralisation test.
INTRODUCTION
Equine herpesvirus 1 (EHV1) is the causative agent of respiratory infection,
abortions, perinatal foal mortality and paralysis in horses. It is responsible for
considerable economic loss to the horse industry world-wide (1, 7). For the prevention
of EHV1 infection in horses, Pneumabort-K vaccine containing inactivated
EHV1 antigen has been reported to be effective, suggesting suitability for field
applications ( 1 , 3). However, the ability of this vaccine to protect against simultaneous
challenge with both EHV1 and EHV4 has been questioned by others, as it may provide
only partial protection (6, 19).
The immunity of horses to E H V I infection is characteristically short-lived and
the animals become susceptible to reinfection within three to four months of recovery,
especially after respiratory infection. Despite the presence of neutralising antibodies
* Department of Veterinary Microbiology, Haryana Agricultural University, Hisar 125004, India.
902
to EHV1 in the sera of animals following vaccination, the animals showed clinical
manifestations (5, 8). It is obvious from the literature that immune mechanisms other
than neutralisation by antibodies are important in protection from E H V 1 . The aim
of the present study was to examine the humoral and cellular immune responses in
donkey mares (the most closely related species to horses) upon primary and secondary
immunisation with live virus and inactivated vaccine, to understand the nature of
short lived immunity to E H V 1 .
MATERIALS AND METHODS
Virus
A local isolate of EHV1 adapted in ovine kidney cell culture containing
approximately 10 T C I D / m l was used for immunisation. Before use as an antigen
in the leucocyte migration inhibition test (LMIT), the virus was inactivated by exposure
to ultraviolet (UV) radiation.
6
5 0
Vaccine
Pneumabort-K vaccine was used for immunisation of the animals following the
recommendations of the manufacturer, i.e. 2 ml intramuscularly (i.m.).
Experimental animals
Apparently healthy non-pregnant donkey mares aged one to two years were
purchased from a local contractor and maintained on gram fodder under conventional
conditions.
Immunisation protocol
Three donkey mares in one group were immunised with Pneumabort-K vaccine
and three in a second group with ovine kidney-adapted live virus (2 ml, i.m.). The
two groups were kept separate from each other. Two donkey mares were maintained
as unvaccinated controls and housed separately. After an interval of three months,
the two groups of donkey mares were given a second immunisation with the same
vaccine using the identical route and dose.
The donkey mares were bled at intervals after primary and secondary immunisation
to obtain serum and leucocytes for virus neutralisation (VN) tests and LMIT, respectively.
Virus neutralisation test
The VN test was performed according to the protocol described by Mitchell and
colleagues (17). Briefly, heat-inactivated serum was diluted two-fold and incubated
with 100 T C I D for 1 h at 37°C. The serum and virus mixture was inoculated into
ovine kidney culture tubes (at least three tubes per dilution of serum) and observed
for 7 days. From these results, VN antibody titres of the sera were determined.
5 0
Leucocyte migration inhibition test
LMIT was performed using (he method previously described (10, 23). The non
toxic (optimal) dilution was deter mined for the UV-inactivated virus harvested from
ovine kidney cell culture and used as antigen; this was found to be 1:4. Leucocytes
903
were separated from the blood of both immunised and normal donkeys by removing
the erythrocytes with hypotonic shock, using chilled triple glass-distilled water (22).
After washing the leucocytes twice with H a n k ' s balanced salt solution, the leucocytes
were finally reconstituted in medium 199 containing 10°7o precolostral calf serum and
antibiotics. The viability of these leucocytes was checked by the trypan blue dye
exclusion technique and found to be >95°7o. Migration of the leucocytes charged
in capillary tubes was tested both with and without the antigen. Four capillary tubes
were used on average per sample and the appropriate controls were included in the
test. The percent leucocyte migration inhibition (LMI) was calculated using the
following formula:
percent LMI
100
area of migration of
immune cells with antigen
—
x 100
area of migration of
immune cells without antigen
:
area of migration of
control cells with antigen
—
— x 100
area of migration of
control cells without antigen
:
RESULTS
Primary and secondary virus neutralisation antibody response
VN tests were carried out on the sera taken at 0, 7, 14, 21, 28 and 90 days after
primary immunisation and 4, 8 and 12 days after the secondary immunisation. After
primary immunisation with the Pneumabort-K or live virus, VN antibody titre
averaged 64, rising to 256 after secondary immunisation. The ability of the two
immunogens to elicit VN antibody responses after primary and secondary
immunisation was observed to be almost identical (Table I).
TABLE I
Virus neutralising antibody titres * of donkey
after primary and secondary immunisation
Days postimmunisation
mares
**
Pneumabort-K
vaccine
Ovine kidney-adapted
strain of EHV1
Non-immunised
control
Primary
0
7
14
21
28
90
1:8
1:64
1:64
1:64
1:128
1:64
1:8
1:64
1:64
1:64
1:64
1:128
1:8
ND
ND
1:8
ND
ND
Secondary
4
8
12
1:256
1:256
1:256
1:256
1:256
1:512
1:8
ND
ND
* reciprocal of highest dilution of serum which neutralised 50% CID50
** the test was carried o u t with pooled sera o b t a i n e d from three animal
N D . no d a t a
904
Primary and secondary leucocyte migration inhibition response
Migration inhibition of peripheral leucocytes was studied at 4, 8, 12 and 16 days
and 4, 8, 10 and 12 weeks after primary immunisation, and 4, 8, 12 and 20 days after
secondary immunisation (Fig. 1). LMI response at four days after primary
immunisation was 44.10% and 41.66% respectively in the two groups, increasing
gradually to 90% at 16 days and then persisting. After secondary immunisation, the
LMI values were 77.30% on day 4, rapidly declining to 2 0 % by day 12. Results were
similar in the two groups, showing a rapid and enhanced response immediately after
secondary immunisation. However, the duration of response was significantly short
compared to the response observed after primary immunisation, indicating a
suppression of LMI response after secondary immunisation.
Time after immunisation
immunisation with P n e u m a b o n - K vaccine
immunisation with ovine kidney adapted strain of E H V ]
Values shown represent the mean of three donkeys per g i o u p , and for each test a duplicate set
of capillarry tubes was used
FIG. 1
Primary and secondary leucocyte migration inhibition
responses of donkey mares immunised with Pneumabort-K vaccine
and an ovine kidney-adapted strain of EHV1
DISCUSSION
Considering the characteristically short-lived immunity to EHV1 vaccine an
immunisation protocol which stimulates an immune response comparable to that
resulting from natural infection would be highly desirable
905
In the present study, the kinetics of the response to inactivated and live virus vaccines
was found to be similar, with VN titres of 1:128 after primary immunisation and 1:256
after secondary immunisation. Intranasal inoculation of pregnant mares with EHV1 and
vaccination with a killed (Pneumabort K) vaccine which produces a threshold level of
VN antibody ( > 1:80) is claimed to correlate resistance to reinfection (2, 4, 18). However,
the occurrence of foetal infection and abortion despite high levels of VN antibodies
suggests that protective mechanisms other than antibody were involved (12, 13).
Cell-mediated immunity (CMI) responses could be detected by L M I T as early as
four days after primary immunisation, reaching a peak of 9 0 % by 16 days. After
secondary immunisation, LMI response was > 7 0 % but decreased rapidly to <20°7o
by 12 days. The C M I responses to the two immunogens were also almost identical
(Fig. 1). It has been observed in certain vaccine trials that some ponies were protected
against infection in the absence of strong concentrations of VN antibodies (19) and
CMI has been reported to play an important role in protection against EHV1 (10, 23).
If the CMI response is important for protection, then a low a n d / o r unpredictable
CMI response after repeated vaccination could be one possible reason for the failure
or breakdown of E H V 1 vaccination.
The suppressed (weak and short-lived) LMI responses after secondary
immunisation noted in this study could have several explanations. Firstly, T cell
responses have been shown to be inhibited by a normal humoral response due to the
formation of antigen-antibody complexes (11). Secondly, there could be impairment
in the induction of certain cytokines, such as Interleukin-2, which regulate CMI (16).
Alternatively, the induction of suppressor cells during primary immunisation may
be responsible for the suppression observed after secondary immunisation (9, 14).
Many other viruses, including other herpesviruses of man and animals, are also
known to be immunosuppressive, working at different levels by various mechanisms
(15, 20, 21). However, the present study is particularly interesting in that although
the primary response was normal, the suppression was observed only after secondary
immunisation of the animals. This calls for a careful re-evaluation of the
recommendations of the manufacturers to use vaccine at frequent intervals during
the entire period of pregnancy against a background of frequent natural exposure
due to endemic E H V 1 . This is all the more true if the major role in achieving the
objective of protection is played by CMI. Further research is in progress, using inbred
laboratory animal models (hamsters and mice) to elucidate the exact mechanism of
EHV1 mediated immunosuppression and its role in predisposing animals to reinfection
with E H V I and other infectious agents.
ACKNOWLEDGEMENTS
Dr M. Singh was supported by the Indian Council of Agricultural Research, New
Delhi, in the form of a Junior Research Fellowship. Dr S. Charan received a research
scientist grant from the University Grants Commission, New Delhi. Assistance offered
by Mr Rajpat and M r K. Parsad is thankfully acknowledged. The authors are also
thankful to the Head of the Department of Veterinary Microbiology at Haryana
Agricultural University for providing the necessary facilities and to Mr R.K. Singla
for typing the manuscript.
906
CINÉTIQUE DE LA RÉPONSE IMMUNITAIRE DES ÂNESSES A L'HERPÈSVIRUS 1
DES ÉQUIDÉS : SUPPRESSION DE LA RÉPONSE A MÉDIATION CELLULAIRE
SECONDAIRE. - M. Singh et S. Charan.
Résumé : Dans le but d'étudier la cinétique de la réponse immunitaire à
l'herpèsvirus 1 des équidés (HVIE), des ânesses ont été immunisées par une
souche de laboratoire de ce virus ou par le vaccin Pneumabort-K aux doses
recommandées (vaccin à adjuvant huileux, préparé à partir de la souche 183
inactivée par le formaldéhyde), avec un rappel trois mois plus tard.
La réponse immunitaire humorale a été mesurée par une épreuve de
neutralisation virale et la réponse cellulaire par un test d'inhibition de la
migration des leucocytes.
Un titre d'anticorps de 1:64 ou 1:128 a été atteint après la première
immunisation et une augmentation marginale (1:256) après la seconde
immunisation par l'un ou l'autre des immunogènes.
Après administration de la première dose d'immunogène, on a pu observer
une augmentation progressive de la réponse immunitaire à médiation cellulaire
de l'hôte, qui a persisté jusqu'à trois mois. Après la seconde immunisation,
cette réponse était par contre de courte durée et peu marquée, comparativement
à la réponse primaire aux deux 'immunogènes. Ce phénomène pourrait expliquer
la rupture de l'immunité anti-HVIE qui provoque des avortements chez les
juments immunisées.
MOTS CLÉS : Epreuve de neutralisation virale - Epreuve d'inhibition de la
migration des leucocytes Herpèsvirus des équidés Immunosuppression
Réponse immunitaire - Réponses immunitaires primaire et secondaire.
INMUNOCINÉTICA DEL HERPESVIRUS EQUINO 1 EN BURRAS: SUPRESIÓN DE LA
RESPUESTA SECUNDARIA MEDIADA POR CÉLULAS. - M. Singh y S. Charan.
Resumen: Para estudiar la inmunocinética del herpesvirus equino 1 (EHV1),
se inmunizaron unas burras con una cepa de laboratorio de EHV1 o con las
dosis recomendadas de vacuna Pneumabort K (cepa EHV1 Ejército 183,
atenuada por formalina, con adyuvante oleoso) y se revacunaron tres meses
después.
Se estudiaron las inmunorrespuestas humorales empleando una prueba de
neutralización del virus (NV) y se utilizó una prueba de inhibición de migración
de los leucocitos para la determinación de las inmunorrespuestas celulares.
Después de la inmunización primaria, el título del anticueipo NV alcanzó
1:64 ó 1:128y mostró un aumento marginal (1:256) después de la inmunización
secundaria con cualquiera de los inmunógenos.
Se comprobó un aumento gradual en la inmunorrespuesta celular después
de la prunela dosis de inmunógeno que persistió hasta tres meses. No obstante,
en la inmunización secundaria, la inmunoirespuesta celular fue de corta duración
y débil en comparación con la respuesta primaria con ambos inmunógenos. Esta
podría ser una explicación posible del fiacaso de la inmunidad anti-EHVl
conducente al aborto en hembras inmunizadas.
907
PALABRAS CLAVE: Herpesvirus equino - Inmunosupresión Prueba de
inhibición de la migración leucocitaria - Prueba de neutralización del virus
Respuesta inmune Respuestas inmunes primaria y secundaria.
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