Immunogenic and protective properties of haemagglutinin protein (H) of Rinderpest virus expressed by a recombinant baculovirus

Vaccine,Vol. 15, No. 6i7, pp. 603-607, 1997 0 1997 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0264410)(197 $17~0.00

Elsevier ELSEVIER

Immunogenic and protective properties of haemagglutinin protein (H) of Rinderpest virus expressed by a recombinant baculovirus Sangeeta

Naik*1_, G.J. Renukaradhyat,

M. Rajasekharl

and M.S. Shaila*§

The hemagglutinin (H) protein of Rinderpest virus expressed by a recombinant baculovirus used as a vaccine produced high titres of neutralizing antibody to Rinderpest virus in the vaccinated cattle, comparable to the levels produced by live attenuated vaccine. The immunized cattle were protected against a vaccine-virus challenge, as demonstrated by the failure of development of antibodies to N protein of the vaccine virus. The lack of replication of vaccine virus in the immunized cattle indicated that they are capable of showing a protective response tf challenged with a virulent virus. 0 1997 Elsevier Science Ltd. Keywords:

haemagglutinin;

Rinderpest

virus; recombinent

baculovirus;

Rinderpest is a devastating disease of cattle caused by the Rinderpest virus (RPV), which is a member of the Morbillivirus genus of the family Paramyxoviridae’. RPV is an enveloped virus; the host cell derived membrane envelope carries two virus specific glycoproteinsthe Hemagglutinin (H) and the Fusion (F) glycoproteins. Immune responses to both the H and the F antigens of the morbilliviruses play an important role in the prevention of infection and attenuated live vaccines have been used to provide protective immunity against this group of viruses. Rinderpest is prevalent in many parts of the developing world and eradication campaigns are at present underway in Africa, the Middle East and South Asia. Today, the only effective time tested vaccine to combat rinderpest is the Plowright’s live attenuated, tissue culture-adapted strain of RPV* which confers lifelong immunity with a single vaccination. Inspite of this, rinderpest remains one of the most devastating and economically important diseases of cattle. The difficulty of maintaining the cold chain is a major cause of vaccine failure since the virus is very heat labile. For the global eradication campaign against rinderpest there is a requirement for a heat stable, effective vaccine and availability of large quantity of rinderpest antigen for subsequent sero-monitoring works. Attempts are being made towards the develop*Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560 012, India. TPresent address: Bioinformatics Centre, University of Pune, Pune. 411 007, India. SICAR project on Animal Disease Monitoring and Surveillance, Institute for Animal Health and Veterinary Biologicals Campus, Hebbal, Bangalore, 560 024, India. §To whom correspondence should be addressed. (Received 25 June 1996; revised 14 October 1996; accepted 4 November 1996)

protective

immunity

ment of heat stable rinderpest vaccines. These include the thermostable Vero cell-adapted rinderpest vaccine”, recombinant rinderpest vaccines using vaccinia virus&” or capripox virus”.‘*. We have expressed and characterized a membrane bound and a soluble form of RPV H protein in a baculovirus expression system”. In this communication, we report the immunogenic and protective properties of the membrane bound form of H protein of RPV expressed by a recombinant baculovirus. MATERIALS

AND

METHODS

Cells and recombinant vaccine Vero (African green monkey) cell line was obtained from the National Institute of Virology, Pune, and grown in milk dilution or Roux bottles in minimal essential medium (MEM) supplemented with 4% foetal or new born calf serum, 100 pug ml-’ penicillin and 100 U mll’ streptomycin. RPV(RBOK) vaccine strain was obtained from the Institute of Animal Health and Veterinary Biologicals, Bangalore, India. Spodoptera fiugiperda (Sf)-9 cells, wild type baculovirus (AcNPV) and recombinant H baculovirus, harbouring the membrane-bound form of RPV H glycoprotein [recH(M)] were maintained as described before”. This recombinant baculovirus expresses a membrane bound form of the RPV H glycoprotein under the influence of the polyhedrin promoter. Briefly, for the production of recombinant vaccine, Sf9 cells at 5060% confluency were infected at a multiplicity of 20 with the recombinant virus recH(M) or A~sNH3.41~~ harvested between 48-72 h when 60-70% cytopathic effect (CPE) was observed. Cells were gently washed once with phosphate buffered saline (PBS) and then resuspended in PBS at 1 x 10’ cells ml- ’ . The suspension

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Immunogenic and protective properties of baculo-H: S. Naik et al. was sonicated in a vibracell (USA) ultrasonic processor with a probe dipping into the solution for 30 s each, three times (40% output), then thoroughly mixed with equal volume of Freund’s complete adjuvant. The monoclonal antibody against RPV N protein has been described previously . Immunization of cattle A group of 15 cross bred cattle (Holstein Friesian X and Jersey X) located on a isolated private farm “Lumbini”, 30 km from Bangalore, were used for this trial. These cattle were free of rinderpest specific antibodies and hitherto were not vaccinated. A group of 10 cattle (the test group) were given 1.2 ml of this baculo-H vaccine at two sites intramuscularly (0.6 ml each site). Another group of five cattle, which formed the control group were given one dose of conventional RPV vaccine (TCRV RBOK) produced at the Institute of Animal Health and Veterinary Biologicals, Hebbal, Bangalore, India, 30th April 1993 batch) intramuscularly. Blood samples were collected before and at various time-points up to 6 months after vaccination. After 20 months, three of the baculo-H vaccinated animals were challenged with one dose each of RPV (RBOK) vaccine (obtained from Institute for Animal Health, Pirbright, UK). Blood was collected after 2, 3.5, 6 and 16 weeks of challenge, in order to assess the extent of multiplication of challenge virus in these immune animals by monitoring the presence of antibodies to N protein. This is an indication of the immune status of the animals. At the same time, three unvaccinated cattle were vaccinated with one dose each of RPV (RBOK vaccine). Blood was collected after 2, 3.5 and 16 weeks to check for the presence of antibodies to N protein. Antibody assays Virus neutralization test. Sera collected at various time points were tested in duplicate in microneutralization test for antibodies against RPV. The assay was performed in flat bottomed 96-well plates as described by Barrett et al6 Briefly, serum samples were inactivated at 56°C for 30 min and then double diluted with culture medium, starting with an initial dilution of 1:lO. To 25 ~1 of every serum dilution placed in separate wells, 100 TCID,, units of RPV(RBOK) in 25 ~1 volume was added. The plate was incubated at 4°C overnight and then l-2 x lo4 trypsinized Vero cells were added to all the wells. Appropriate controls were included in every plate. The plate was incubated at 37”C, 5% CO, till 80% CPE was seen in virus control wells (4-5 days). The plate was then read under the microscope and the CPE estimated visually. Estimation of antiH and antiN antibodies by competition ELISA Sera collected from the baculo-H vaccinated cattle and the control group were tested for the presence of antiH antibody by the method of Anderson and McKay13, using a monoclonal antibody to RPV H protein (kind gift from Dr T. Barrett, Institute for Animal Health, Pirbright, UK). The presence of antibodies specific for N protein was monitored in the sera of the three immunized cattle

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Figure 1 AntiH antibody levels in cattle immunized by baculo-H recombinant protein by competition ELISA using monoclonal antibody against H. Broken line indicates 50% inhibition, any value above it is considered significant and positive for presence of rinderpest antibodies

challenged with vaccine virus by a competitive ELISA with a monoclonal antibody to Nr2 using purified RPV as anti en employing the method of Anderson and McKay 91 -. Immunoperoxidase staining of RPV infected Vero cells with cattle sera A 1.5 ml sample of MEM was added to 35 mm dishes and then sterile coverslips were placed on them. Trypsinized Vero cells (5-6 x 106) were added to each dish and the dish gently swirled for even distribution. Dishes were incubated at 37°C 5% CO? for about 24-36 h, till they were about 40% con&tent. Cells were then infected with RPV at a multiplicity of 0.1-0.5 and incubated at 37°C for 48-72 h till 30-40% CPE was seen. The medium was discarded, coverslips were washed once with PBS and then subjected to immunoperoxidase staining according to Harlow and Lane14.

RESULTS After vaccination with either baculo-H recombinant antigen or tissue cultured rinderpest vaccine (TCRV), all animals remained healthy and did not develop any reaction at the vaccination site. One of the baculo-H vaccinated cow (ClO) died during the course of the trial due to an unrelated cause and therefore was not considered further. Rinderpest specific antibodies detected in cattle sera by competition ELISA using rinderpest antigen and rinderpest monoclonal antibody (aH) are shown in Figure 1. Seroconversion (50% inhibition) of baculo-H vaccinated cattle was observed as early as 1 week postvaccination. Peak levels of antibodies were observed between 8-12 weeks and a value as high as 81% was observed. Cattle remained positive for rinderpest antibodies at least up to 22 weeks after immunization. The virus neutralizing (VN) antibody response is shown in Table I. A two to eightfold increase in the VN

Immunogenic and protective properties of baculo-H: S. Naik et al. Table 1 of RPV

Virus

neutralization

Weeks Animal

No.

0

Test group: baculo-H Cl I:

titres of antibodies

RPV

after vaccination

of cattle

with recombinant

baculovirus

expressed

H protein

post-vaccination 1

2

3

4

8

12

16

22

160 80 640 160 20 160 160

320 40 160 80 160 160 cl0 80 160

160 N.D. 160 :0 80 N.D. 160 160

>1280 1280 1280 >1280 1280 160 640 EO

640 >1280 >1280 >1280 >1280 320 640 1280 >1280

1280 >1280 N.D. >1280 >1280 >1280 S-1 280 N.D. >1280

1280 N.D. N.D. >1280 z-1280 >1280 >1280 N.D. >1280

80 40 180 80 160

80 40 160 80 160

320 80 160 160 >1280

640 1280 640 1280 640

>1280 N.D. 1280 >1280 >1280

>1280 >1280 >1280 640 N.D.

>1280 >1280 1280 1280 N.D.

vaccinated 80

:i

:5

80

N.D. 160

:“s C6

:: cl0

80 160 10

z c9

cl0 20 10

20 160 80

Control group: TCRV vaccinated Cl1 :: :: Cl2 Cl3 80 :: Cl4 10 Cl5
against

68

Vaccine Virus challenge

Not determined

titre was observed in the baculo-H vaccinated cattle sera as early as 1 week after immunization. By 8-12 weeks post-immunization, all the test group cattle had very high titres of VN antibodies which persisted up to 22 weeks after immunization. The VNT titres of baculo-H vaccinated cattle were comparable with those of TCRV vaccinated cattle. The antibodies were shown to react with the virus antigen made in infected cells by immunoperoxidase staining (Figure 2) thus demonstrating its specificity against virally coded protein. Positive staining reaction was observed 4 and 8 weeks post-vaccination in the baculo-H vaccinated (Figure 2e and g) cattle and its degree was stronger than the preimmune or the control group. Twenty months post-baculo-H vaccination, three of the baculo-H vaccinated cattle were challenged with the RPV(RBOK) vaccine virus, and tested for the presence of antiN antibodies by competition ELISA at 2, 3.5 and 16 weeks post-challenge. No antiN antibodies were detected indicating that the RPV(RBOK) vaccine virus had failed to replicate in these cattle. In the three unvaccinated positive control animals which received the “RBOK vaccine virus” challenge, the antibodies against N protein was detected at 2, 3.5 and 16 weeks after challenge. The vaccine virus was used as challenge instead of a virulent virus due to lack of disease security facility. Under these circumstances, the absence of antibodies to a major protein of the virus is a measure of the inability of the challenge vaccine virus to replicate in the animals which received recombinant vaccine.

DISCUSSION The H protein of morbilliviruses is responsible for attachment of the virus to the host cell receptor and elicits significant level of neutralizing antibodies’5*‘6. It is most variable of-the envelope associated proteins and may be responsible for the different host tropisms of the individual morbilliviruses. Most of the other virus proteins including the two membrane associated proteins F and the matrix (M) proteins are highly conserved across pox viruses expressing the H the group I7. Recombinant

protein of RPV alone have been shown to confer immunity to the disease4*“*“. We have expressed the membrane bound and soluble forms of RPV H protein in a baculovirus expression system”. The immunogenic properties of the membrane bound form have been tested in protection-challenge experiments in rabbits (data not shown). When crude cell extract of recombinant virus infected cells were used as immunogen, partial protection was observed when the rabbits were challenged with lapinized virus. Partially purified membrane fraction of recombinant virus infected cells when used as an immunogen, conferred complete protection in rabbits. This prompted us to test the immunogenic properties of the recombinant protein in cattle. This consisted of a one shot immunization followed by seromonitoring up to 6 months. Protection challenge trials with virulent RPV could not be conducted in cattle due to lack of high containment facilities. Instead, three of the baculo-H vaccinated cattle were challenged with RPV(RBOK) vaccine virus 20 months after immunization and tested for the presence of antiN (nucleocapsid protein) antibody, which would give an indication for RPV replication. Immunogenic response was assessed by monitoring antibody levels and in vitro protection assay in terms of virus neutralization. Serum neutralizing antibodies were detected as early as 1 week after vaccination of cattle with baculo-H recombinant protein. All baculo-H vaccinated cattle developed high titres of SN antibodies (1280) by 8-12 weeks post-vaccination and these levels remained high up to 22 weeks (Table I). Specific antibodies were also detected by cell staining of RPV infected Vero cells. Three of these baculo-H vaccinated cattle were challenged with RPV(RBOK) vaccine virus 20 weeks postvaccination and assessed for the multiplication of the vaccine virus by monitoring for the presence of antiN antibodies. No antiN antibodies could be detected in the sera of these challenged cattle up to 16 weeks postchallenge, indicating complete absence of the vaccine virus replication in vivo. Thus the baculo-H recombinant vaccine appears to have a potential to confer protective immunity against rinderpest.

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Immunogenic and protective properties of baculo-H: S. Naik et al.

staining of RPV infected Vero cells by sera obtained from baculo-H vaccinated cattle. (a) Positive control strum Figure 2 lmmunoperoxidase (b), (d) and (f) TCRV vaccine cattle, (c), (c) and (g) baculo-H vaccinated cattle, (b) and (c) preimmune, (d) and (e) 4 weeks and (f) and (g) 8 weeks after immunization. Vero cells grown on coverslips were infected with RPV(RBOK) at a moi of 0.1-0.5 and incubated for 48-72 h till 30-40% CPE is seen. Medium was discarded and after washing once in PBS. the coverslip cultures were fixed for immunoperoxidase staining Bassiri et ~1.” have reported that the expression of RPV H and F in the baculovirus expression system did not confer protection. These negative results could have been due to the use of low amounts of antigen, presence of contaminating proteins, and Freund’s incomplete adjuvant. Although Freund’s complete adjuvant, used in the present study, is not a desirable adjuvant for vaccination purpose, results obtained here do indicate that the baculo-H recombinant vaccine is immunogenic in cattle and produced a protective immune response. Taken together these results indicate the need for a

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standard experimental protocol with respect to amount or dose of antigen and the use of a proper adjuvant as key features to elicit a good immune response.

ACKNOWLEDGEMENTS We the out by

thank Dr Ravi Reddy of Lumbini Farm for all help and cooperation extended to us for carrying cattle immunization work. The expert help given the farm veterinarian, Dr Murali Pai is gratefully

Immunogenic and protective properties of baculo-H: S. Naik et al. acknowledged. We thank Mrs Madhuri P. Apte for her expert technical help. This work is funded by the Department of Biotechnology, Government of India, under the molecular virology programme. One of us (SSN) thanks the Council of Scientific and Industrial Research, Government of India, for a Senior Research Fellowship.

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Yamanouchi, K., Inui, K. and Sugimoto, M. et al. lmmunisation of cattle with a recombinant vaccinia vector expressing the hemagglutinin gene of rinderpest virus. Vet. Rec. 1993, 132, 152-l 56 11 Romero, C.H., Barrett, T. and Evans, S.A. et al. Single capripox virus recombinant vaccine for the protection of cattle against rinderpest and lumpy skin disease. Vaccine 1993, 11, 737-742 12 Bhavani, K., Karande, A.A. and Shaila, M.S. Preparation and characterisation of monoclonal antibodies to nucleocapsid protein N and H glycoprotein of rinderpest virus. Virus Res. 1989, 12, 331-348 13 Anderson, J. and McKay, J.A. The detection of antibodies against peste des petits ruminants virus in cattle, sheep and goats and the possible implication to rinderpest control proaramme. kidemiol. Infect. 1994, 112, 225-321 14 Harlow, E.’ and Lane, D. (Eds). Antibodies-A Laboratory Mama/. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1988, p. 402 15 Choppin, P.W. and Schied, A. The role of viral glycoproteins in adsorption penetration and pathogenicity of viruses. Rev. Infect. Dis. 1980, 2, 40-61 16 Varsanyi, B., Morein, B., Love, A. and Norrby, E. Protection aaainst lethal measles virus infection in mice by Immune scmulating complexes containing the haemagglutinin or fusion orotein. J. l&o/. 1987, 61, 3896-3901 17 ‘Harder, T.C., Klusmeyer, i., Frey, HR., Orvell, C. and Liess, B. Intertypic differentiation and detection of intratypic variants among canine and phocine morbillivirus isolates by kinetic neutralisation using a novel immunoplaque assay. J. viral. Met. 1993, 41,77-92 18 Romero, C.H., Barrett, T., Chamberlain, R.W.. Kitching, R.P., Fleming, M. and Black, D.N. Recombinant capripox expressing the hemagglutinin protein gene of rinderpest virus-protection of cattle against rinderpest and lumpy skin disease viruses. Virology 1994, 204, 425-429 19 Sangeeta, N. and Shaila, MS. Characterization of membranebound and membrane anchor-less forms of hemagglutinin glycoprotein of Rinderpest virus expressed by baculovirus recombinants. Virus Genes 1997 (In Press) 20 Bassiri, M., Ahmad, S., Giavedoni, L., Saliki, J.T., Mebus, C. and Yilma, T. Immunological responses of mice and cattle to baculovirus expressed F and H proteins of rinderpest virus: lack of protection in the presence of neutralising antibody. J. Viral. 1993, 67, 1255-1261

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