Rinderpest virus (RPV) ISCOM vaccine induces protection in cattle against virulent RPV challenge

Vaccine 19 (2001) 3355– 3359 www.elsevier.com/locate/vaccine

Short communication

Rinderpest virus (RPV) ISCOM vaccine induces protection in cattle against virulent RPV challenge Hiroshi Kamata a,1, Kazue Ohishi a,2, Ellena Hulskotte b, Albert D.M.E. Osterhaus b, Kenjiro Inui a, M.S. Shaila c, Kazuya Yamanouchi d, Thomas Barrett a,* a

Institute for Animal Health, Pirbright Laboratory, Ash Road, Woking, Surrey GU24 0NF, UK b Institute for Virology, Erasmus Uni6ersity, Rotterdam, The Netherlands c Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India d Nippon Institute for Biological Science, 9 -2221 -1, Ome, Tokyo, Japan

Received 17 October 2000; received in revised form 6 February 2001; accepted 8 February 2001

Abstract Rinderpest 6irus (RPV), a member of genus Morbilli6irus in the family Paramyxo6iridae, causes an acute and often fatal disease in cattle and other large ruminants. A subunit rinderpest vaccine consisting of an immune-stimulating complex (ISCOM) incorporating the RPV haemaggulutinin (H) protein, was examined for its ability to induce protective immunity in cattle, the natural host of RPV. All of four cattle vaccinated with the ISCOM vaccine survived challenge with virulent virus. Three were solidly protected, showing no clinical signs of infection, while the fourth animal developed only mild and transient symptoms. Virus neutralizing antibodies were produced at a significant level in all vaccinated cattle. These results indicate that this ISCOM vaccine is effective in producing protective immunity in cattle and should be a suitable means of delivering glycoprotein antigens from other morbilliviruses. © 2001 Elsevier Science Ltd. All rights reserved.

Rinderpest 6irus (RPV) is a member of the genus Morbilli6irus, and closely related to causative agents of measles in humans and distempers in dogs, seals and cetacean species [1]. It is responsible for an economically important disease affecting cattle and wild ruminants. Poxvirus-based vector vaccines expressing RPV haemagglutinin (H) or fusion proteins have been developed which protect cattle against a lethal RPV challenge [2–7]. The involvement of cell-mediated immunity in the protection by these vector vaccines was suggested [8 – 10]. In spite of the efficacy of vaccinia-vectored vaccines, possible adverse reactions in humans accidentally in-

* Corresponding author. Tel.: + 44-1483-232441; fax: + 44-1483232448. 1 Present address: Laboratory of Veterinary Microbiology, Nihon University, Fujisawa, Japan. 2 Present address: Otsuchi Marine Research Centre, Ocean Research Institute, University of Tokyo, Japan.

fected with the recombinant vaccines have made their use highly controversial [11]. In this respect, subunit vaccines consisting of only purified viral proteins, are much safer, however, they normally only induce a humoral antibody response and this is a major drawback since neutralizing antibody alone cannot protect against morbillivirus infection. Baculovirus expressed H and F proteins of RPV failed to induce protective immunity despite having stimulated a strong neutralizing antibody response in vaccinated cattle [12]. Immune-stimulating complex (ISCOM) systems incorporating viral proteins have been shown to induce both humoral and cell-mediated immunity, including cytotoxic T-cell responses, which can protect against subsequent challenge [13 –15]. We constructed an ISCOM vaccine incorporating only the RPV H protein, purified from cells infected with a baculovirus recombinant expressing this protein [16], and examined its efficacy in protection of cattle from lethal challenge with RPV.

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H. Kamata et al. / Vaccine 19 (2001) 3355–3359

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Fig. 1. Experimental design of vaccination and challenge. Arrows indicate the days of the two ISCOM vaccinations and the day of challenge with the virulent Saudi1/81 strain of RPV.

The recombinant baculovirus expressed the RPV H protein as a membrane-bound protein in infected Sf21 insect cells and the protein was purified by solubilizing purified cell membranes with octylglycoside. Briefly, Sf21 cells infected with this recombinant (moi 5) were washed with Ca– Mg-free phosphate buffered saline (PBS) at 48 h post infection and resuspended in a buffer consisting of Ca–Mg free PBS, 10% sucrose, 2 mM EDTA, 1 mM PMSF, and then kept on ice for 5 –10 min. The suspension was centrifuged at 22000 rpm for 1 h in a Beckman SW50 rotor. The pellet was resuspended in hypotonic buffer (50 mM phosphate buffer, pH 7.4 containing 3 mM PMSF) and centrifuged again at 14000 rpm for 30 min in the same rotor. The pellet was finally resuspended in lysis buffer (PBS containing 1.5% octylglucoside and 2 mM PMSF) and kept on ice for 1 h. The suspension was centrifuged at 35000 rpm for 1 h in an SW50 rotor to remove non-solubilized debris and the supernatant, after dialysis against PBS, was used for vaccine preparation. ISCOMs incorporat-

ing the RPV H protein were produced according to a standard method [17]. The vaccination and challenge protocol is shown in Fig. 1. Four Friesian cross Aberdeen Angus calves (SR89, SR91, TA25 and TA26) were inoculated subcutaneously with the ISCOM vaccine containing 100 mg RPV H protein in a volume of 1.0 ml. After 5 (SR89 and SR91) or 6 (TA25 and TA26) weeks, the cattle received the second vaccination with the ISCOM vaccine incorporating 50 mg RPV H protein. Three cattle (SR24, SR25 and SR26) were used as unvaccinated controls. All seven animals were challenged with 104TCID50 of virulent Saudi 1/81 strain of RPV [18] at 25 weeks (SR89 and SR91) or 15 weeks (TA25 and TA26) after the first vaccination. Following challenge rectal temperatures were recorded daily and the cattle were examined for clinical signs of rinderpest infection. The three unvaccinated controls developed severe clinical signs of rinderpest, high fever, severe stomatitis and diarrhea, and were euthanized on either day 9 or 10 following challenge. In contrast, all four vaccinated cattle survived the challenge. Three of the four vaccinated cattle were solidly protected from the disease and showed no clinical signs of infection throughout the experiment. The remaining animal (SR91) developed a delayed and transient fever and a mild mouth erosion but quickly recovered. Uncoagulated blood was collected on days 3, 5, 7 and 14, and the leukocytes were counted to check for leukopenia, a sign of morbillivirus replication. Severe leukopenia was observed in all control animals on days 5 and 7. A similar severe leukopenia was seen in SR91 on days 5 and 7 but by day 14, the leukocyte count had returned to normal. The development of clinical signs is detailed in Table 1. Virus isolation from peripheral blood leukocytes (PBL) was attempted by co-cultivation with B95a cells,

Table 1 Clinical signs and leukopenia following challenge Cattle

Clinical signsa Feverb

Stomatitis

Diarrhea

Control SR24 SR25 SR26

+(4) +(4) +(4)

++(6) ++(4) ++(4)

+(9) +(9) +(9)

Vaccinated SR89 SR91 TA25 TA26

– +(6) – –

– +(7) – –

a

– – – –

Leukopenia (% decreasec)

Protection

83 86 76

No No No

34 71 10 0

Complete Partial Complete Complete

Numbers in parentheses indicate the day post-challenge of the first appearance of the signs. Cattle showing a temperature greater than 39.5 were taken as positive. c The % decrease was calculated as 100×(number of cells before challenge−smallest number of cells after challenge)/number of cells before challenge. b

H. Kamata et al. / Vaccine 19 (2001) 3355–3359 Table 2 Virus isolation from PBL following challengea Cattle

Viraemia (days post challenge) 0

3

5

7

14

Control SR24 SR25 SR26

– – –

– – –

+ + +

+ + +

Dead Dead Dead

Vaccinated SR89 SR91 TA25 TA26

– – – –

– – – –

– – – –

– + – –

– – – –

a PBL (106) from each animal were co-cultivated with B95a cells (5×105) in each of five wells of a 96-well microtitre plate.

which are highly sensitive hosts for replication of RPV [19]. PBL (106) from each animal were co-cultivated with B95a cells (5× 105) in each of five wells of a 96-well microtitre plate. As shown in Table 2, challenge virus was not detected in the blood from the three cattle that were completely protected, but it was detected on day 7 in the partially protected animal. All the control animals were viraemic on days 5 and 7. The severity of pathognomic clinical signs, leukopenia and viraemia correlated with the degree of protection seen. The development of virus neutralizing antibody was measured in a microneutralization test using Vero cells as earlier described [20]. Two of the four cattle developed significant levels of neutralizing antibody after the first vaccination, while after the second vaccination, high levels of neutralizing antibody were present in all four animals. Following challenge, a slight increase in antibody titers was observed in the completely protected cattle (SR89, TA25, and TA26), whereas a rapid

Fig. 2. Time course of neutralizing antibody responses to RPV in cattle. Arrows indicate the days of the two ISCOM vaccinations and the day of challenge with the virulent Saudi1/81 strain of RPV. The assay was carried out in quadruplicate and the results are given as the log2 titer that gave 50% neutralization.

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rise in antibody titer was seen in the partially protected animal (SR91). The control cattle remained antibodynegative throughout the experiment. The time course of neutralizing antibody response is shown in Fig. 2. In this study, we have shown that ISCOMs incorporating RPV H protein can protect the cattle against challenge with a highly virulent strain of RPV [18]. Complete protection was observed in cattle with higher levels of neutralizing antibody (\ 5 log 2) at the time of challenge, whereas the animal with the lowest titer (5 log 2) was partially protected, showing a delayed and transient fever, mild stomatitis and viraemia. From this it appears that neutralizing antibodies play some role in protection against rinderpest infection, however, cattle inoculated with baculovirus expressed RPV H protein in Freund’s incomplete adjuvant, which preferably induces a humoral immunity, were not protected from challenge, despite high levels of neutralizing antibody [12]. Baculovirus expressed H protein when administered in Freund’s complete adjuvant, which is known to induce both humoral and cellular immunity, could prevent replication of the attenuated tissue culture vaccine in cattle [16], however, no virulent challenge was attempted in that experiment. In any case, Freund’s complete adjuvant is not acceptable for non-experimental animal vaccination. A cell proliferation assay in response to RPV antigen was carried out on PBL from two ISCOM-vaccinated cattle (SR89, SR91). A low but significant (stimulation index of 3.4) response was observed 1 week after the first vaccination in one of the completely protected cattle (SR89) while SR91, which was partially protected did not respond. Although further experiments examining cell-mediated immune responses to the ISCOM-H vaccine are necessary to establish its significance, the rapid increase in the neutralizing antibody titer in the partially protected animal may indicate that primed helper T cells responded to the antigenic stimulus of the challenge virus. In the case of vaccinia and capripox recombinant vaccines expressing the H protein of RPV, the relative importance of cell-mediated immunity over neutralizing antibody responses was suggested by the fact that protection was observed in the absence of detectable neutralizing antibodies [7–9]. These results together strongly suggested that the ISCOM system can stimulate both humoral and cell-mediated immunity in cattle. The degree of protection provided by the ISCOM vaccine appears to be comparable to that given by the vaccinia- and capripoxvirus-based recombinant vaccines. Protection was shown to persist for up to 3 years in some cattle following vaccination with the poxvirus recombinants ([10], Barrett and Wamwayi, unpublished observations). A similar duration of immunity may or may not be found with the ISCOM vaccine, nevertheless, the demonstration of protective immunity against

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challenge with the most virulent strain of RPV indicates the practical usefulness of the ISCOM vaccine for short-term protection in the field. The success of the global rinderpest eradication program (GREP) co-ordinated by the FAO means that most countries where the disease was once endemic have declared provisional freedom from rinderpest and must now cease vaccination with the tissue culture attenuated vaccine since its use masks the presence of mild disease [21,22]. The ISCOM vaccine induces only anti-RPV H antibody, so that it will be compatible with the current serological tests, which are based on the use of a specific monoclonal anti-RPV H antibody in a competitive ELISA [23], for monitoring the effectiveness of vaccination teams. However, by carrying out a second analysis, based on the use of an anti-RPV N antibody, it will also be possible to distinguish vaccinated animals from naturally recovered animals [24]. The former, unlike the latter, will not have developed antibodies to the virus N protein. An ISCOM-adjuvanted vaccine has proved successful in protecting against equine influenza and this vaccine has been licensed for use [25]. Human trials have shown that a formalin-killed influenza vaccine formulated into ISCOM particles was capable of increasing virus-specific CTL memory in 50 – 60% of recipients compared with only 5% of recipients with the standard vaccine, indicating that this adjuvant may be suitable for use in other human vaccines [26]. A global measles eradication campaign, similar to that for rinderpest [21,22], is being supported by the WHO [27]. One major problem encountered in the measles campaign is the lack of suitable vaccine for use in children under 9 months of age when they have varying levels of maternal antibody, which interferes with the response to the live attenuated measles vaccine [28]. Many children in measles endemic countries are infected during this vulnerable period and an ISCOM vaccine could be considered a candidate for a new type of recombinant vaccine, which could confer protection in the face of maternal antibody. Acknowledgements We thank Dr S Ohkubo, Morinaga Milk Industry Co. Ltd., Japan for providing technical advice on the insect cell culture. This research was supported by a grant from the British Council and a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan and Overseas Researcher Grant from Nihon University, Japan. References [1] Barrett T. Rinderpest and distemper viruses. In: Webster RG, Granoff A, editors. Encyclopedia of Virology, vol. 3. London: Academic Press, 1999:1260 –8.

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