Bibliography
T. Yilma
Tilahun Yilma is Professor of Virology of the Department of Veterinary Microbiology and Immunology, Laboratory of Molecular Biology for Tropical Diseases, School of Veterinary Medicine, University of California, Davis, CA 95616, USA.
The candidate (prototype/vaccinia vector rinderpest vaccine, HA and F genes) was developed by Prof. Yilma. The vaccine has undergone preliminary tests but its field value has still to be confirmed. The recombinant vaccine will have a much longer shelf-life which is important for rinderpest campaigns in tropical countries since the Plowright-type RBOK tissue culture preparation applied at present is thermolabile.
Vaccination has been practiced by the peoples of China, Turkey and Africa for hundreds, if not thousands, of years. Modern vaccination, however, began in 1796 when Edward Jenner observed that milkmaids who had been infected with cowpox did not subsequently develop smallpox. He then began to vaccinate people against smallpox by scratching them with scabs from cowpox lesions. Over the past two centuries, this vaccination procedure has been repeated worldwide, using a vaccine strain of poxvirus known as the vaccinia virus. The exact origin of vaccinia virus is unknown. It does not exist in nature and is distinct from cowpox virus and the variola virus of smallpox. Vaccinia virus has protected hundreds of millions of people worldwide from smallpox, with minimal side effects and no adverse environmental impact despite the release of the vaccinia virus on thousands of occasions because of poor hygienic conditions.
A remarkable new generation of vaccines was developed recently, including subunit, synthetic peptide and virus vector vaccines. Recombinant DNA technology has made it possible to insert and express heterologous genes in a variety of different viruses. So far, simian virus 40, bovine papillomavirus, adenovirus and members of the retrovirus family have been used the most extensively as expression vectors. Although the relatively small size of these viruses facilitates genetic engineering, this property also restricts the amount of DNA that can be inserted into the virus particle. In addition, virus vectors usually have a limited host range and are defective, requiring either a helper virus or special cell lines for replication. Furthermore, mans: of these vectors are oncogenic and may therefore be unsuitable for use as vaccines in humans and domestic animals.
One of the most promising new vaccine developments uses infectious vaccinia virus as a vector and produces recombinants through the insertion of genes as protective antigens of disease agents into the vaccinia virus DNA. Vaccinia virus, a member of the poxvirus family, is a large, double-stranded DNA virus that incorporates a complete transcription system and replicates in the cytoplasm of infected cells (Moss, 1974). The virus has a wide host range, including humans, cattle, horses, swine, sheep, goats, mice and monkeys. It also has a large genome which can accept heterologous genes. Recombinant DNA technology has developed convenient marker rescue techniques and plasmid sequences homologous to the virus DNA, thus allowing recombination within infected cells (Mackett et al., 1985). When these recombinant viruses are injected into an animal they replicate in the host cells, expressing both vaccinia virus and foreign genes. The animal thus becomes immune both to vaccinia virus and to the agent from which the foreign gene was taken (see Fig. 1).
1. A schematic representation of a vaccinia virus recombinant that expresses a foreign gene - Schéma d'un recombinant du virus de la vaccine qui exprime un gène étranger - Representación esquemática de un recombinante del virus de la vacuna que expresa un gene extraño
As compared to whole virus vaccines, there are a number of advantages to infectious vaccinia virus recombinant vaccines. Since this system permits the expression of single gene coding for immunogenic proteins, it allows the development of serological tests to differentiate vaccinates from animals infected naturally. Living or killed whole-organism vaccines occasion a full range of immune responses and thus do not allow such distinctions to be made. The inability to differentiate vaccinates from infected animals causes problems in disease diagnosis and control and is responsible for significant economic losses from bans on the import and export of such livestock and their products.
With the infectious vaccinia virus recombinant system, it is possible to make a polyvalent vaccine which expresses several genes representing various serotypes of the same agent or a number of unrelated agents (see Fig. 2). There is also amplification of the antigen, as in the case of live virus vaccines, through DNA replication and transcription. Finally, unlike many live vaccines, the lyophilized form of vaccinia virus is heat stable, obviating the need to maintain the cold chain. This is a major advantage when carrying out a vaccination programme in developing countries.
An infectious vaccinia virus recombinant vaccine, developed recently for rinderpest in the Laboratory of Molecular Biology for Tropical Diseases, illustrates the potential of the vaccines described above. Rinderpest is not only the single most important disease of livestock in developing countries, but it has also played a crucial role in the development of the veterinary profession.
Rinderpest is an acute, febrile, highly contagious viral disease of ruminants, particularly cattle and buffaloes, which manifests itself in a rapid course and high mortality. The disease is characterized by inflammation, haemorrhaging, necrosis and erosion of the gastro-intestinal tract, accompanied by bloody diarrhoea, wasting and death (Plowright, 1968).
The Plowright tissue culture vaccine (PTCV) is widely used for vaccination against rinderpest, despite the difficulty of sustaining manufacture of the vaccine and delivering it to the field; a lack of skilled personnel; a lack of refrigeration; and vaccine instability. On the other hand, the lyophilized form of vaccinia virus is heat stable, easily produced and transported, and is administered by scarification.
The rinderpest virus (RPV) is enveloped and has a single-stranded RNA genome with a minus polarity. The virus belongs to the family Paramyxoviridae and is a member of the morbillivirus group, along with the measles virus of humans, distemper virus of dogs, and peste-des-petits-ruminants virus (PPRV) of goats and sheep. In paramyxoviruses, the haemagglutinin (HA) and fusion (F) surface proteins have been shown to provide protective immunity. The highly virulent Kabete "O" strain of RPV was propagated in primary bovine kidney cells where it characterized eight viral proteins (Grubman et al., 1988). cDNA copies of the HA and F mRNAs were made and the complete nucleotide sequences of both the HA and F genes were determined (Yamanaka et al., 1988; Hsu et al., 1988). Standard procedures were used to construct vaccinia virus recombinants expressing the HA gene (vRVH) and the F gene (vRVF) of RPV (Yilma et al., 1988).
Protective immune response studies in cattle were conducted according to proper institutional guidelines at the high containment facility at the Plum Island Animal Disease Laboratory. Humoral responses of cattle were assessed by serum neutralization (SN) assay. Two separate studies were conducted in animals that were shown to be sero-negative to RPV prior to vaccination. In the first study, two animals were vaccinated with the recombinant vRVH, two with the recombinant vRVF, two with a cocktail of both recombinants (vRVH + vRVF) and two with PTCV as a positive control. Cows were vaccinated intradermally with 108 PFU of vaccinia virus recombinants on day 0 and day 28. They were challenged 42 days after the primary vaccination with a 1 × 1 000 lethal dose of RPV. SN titres are expressed as the reciprocal of the dilution of serum that gave complete protection against the cytopathic effect of 550 TCID50 of RPV (see Table 1). An additional two were left unvaccinated as a negative control. Vaccinia virus recombinants were administered by intradermal inoculation and scarification with 4x103 PFU of virus, and 1 ml of PTCV (106 TCID50) was administered subcutaneously. Four weeks after the primary vaccination, a second dose of recombinant vaccine was administered. Positive controls were not revaccinated with PTCV.
In the second study, only a single vaccination was administered. Cows were vaccinated intradermally with 108 PFU of vaccinia virus recombinants on day 0. Titres are expressed as the reciprocal of the dilution of serum that gave complete protection against the cytopathic effect of 125 TCID50 of RPV (see Table 2). A group of five animals was used for each recombinant (vRVH and vRVF) and for the cocktail of both recombinants (vRVH and vRVF). Two animals were vaccinated with PTCV for a positive control and two were left unvaccinated for a negative control. For a vaccinia virus control, two were vaccinated with v50, a vaccinia virus recombinant expressing the G gene of vesicular stomatitis virus (Mackett et al., 1985).
Pock lesions developed as early as four days in all animals vaccinated with the recombinants, but they were limited to the site of inoculation and had healed completely in two weeks (see Fig.3). All animals vaccinated with the recombinants or PTCV produced SN antibodies to RPV. As expected, control animals had no detectable antibody titres (see Tables l and 2).
To evaluate protection, all animals were challenged with a heavy dose (103 TCID50) of RPV, administered subcutaneously in the prescapular lymph node region. As little as one TCID50 induced clinical rinderpest with 100 percent mortality (data are not shown). In the first study animals were challenged on day 42 and in the second study on day 35, following primary immunization.
The animals vaccinated with the recombinants or PTCV were completely protected from rinderpest, exhibiting no detectable illness and having a normal temperature of 101°F. All controls developed high fever (108°F) by day two after challenge and died by day six. They also developed lesions typical of severe rinderpest, characterized by sloughing and erosion of the epithelial lining of the gastro-intestinal tract and bloody diarrhoea. When no clinical disease was found in vaccinated animals after two weeks of daily monitoring, the experiment was terminated.
Rinderpest is a potential candidate for eradication using the vaccinia virus recombinant vaccine. There is only one serotype of RPV, although there are different strains manifesting different degrees of pathogenicity in the field. A vaccine against one strain will immunize against all, including PPRV of sheep and goats. Because of the close antigenic relationships of the morbilliviruses, the recombinant vaccine for rinderpest may well provide protection against distemper in dogs and measles in humans (Plowright, 1968). The use of vaccinia virus as a vector that can express heterologous viral antigens allows its use as a live virus vaccine for rinderpest, combining the safety advantages of a subunit vaccine with the antigen amplification and native presentation given by a dive attenuated virus vaccine.
Thus, at least for rinderpest, the potential of vaccinia virus recombinant vaccines to protect against a massive challenge dose of virus has been clearly demonstrated. It is important, however, that safety considerations be addressed before introducing live recombinant viruses into the environment. With this in mind, the recombinant vaccines with the attenuated strain, used in successful smallpox eradication campaigns worldwide, were created in the laboratory. Insertional inactivation of the viral TK gene during the process of recombination further attenuates the virus and, at present, investigations are being carried out on the use of lymphokine genes, such as interferongamma or interleukin-2, to enhance the immune response and eliminate all reasonable expectation of risk from the vaccinia virus recombinant vaccines (Yilma et al., 1987; Anderson, Fennie and Yilma, 1988). It was recently demonstrated that expression of interleukin-2 prevented disseminated vaccinia virus infection in immunodeficient mice (Flexner, Hugin and Moss, 1987).
1. SN titres of cattle and response to challenge with RPV
Titres de SN des bovine et réaction a la dose d'épreuve avec le vaccin antibovipestique
Títulos de SN de vacunos y respuesta a la infección con el virus de la peste bovina
|
Vaccine |
Average titres | |||||||
|
|
Pre-challenge |
Post-challenge | ||||||
|
|
Day 0 |
Day 7 |
Day 14 |
Day 21 |
Day 28 |
Day 35 |
Day- 42 |
Day 63 |
|
None |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
Dead |
|
PTCV |
0 |
130 |
374 |
768 |
640 |
640 |
384 |
768 |
|
vRVH |
0 |
48 |
443 |
704 |
389 |
1 280 |
1 786 |
1 792 |
|
vRVH+vRVF |
0 |
16 |
256 |
261 |
384 |
1 152 |
1 408 |
896 |
|
vRVF |
0 |
2 |
8 |
12 |
12 |
56 |
96 |
160 |
2. SN titres of cattle after a single vaccination and challenge with RPV
Titres de SN des bovine après une seule vaccination et dose d'épreuve avec le virus pestique
Títulos de SN de vacunos después de una sola vacunación e infección con virus de la peste bovina
|
Vaccine |
Average titres | |||||||
|
|
Pre-challenge |
Post-challenge | ||||||
|
|
Day 0 |
Day 7 |
Day 14 |
Day 21 |
Day 28 |
Day 35 |
Day- 42 |
Day 63 |
|
None |
- |
- |
- |
- |
- |
- |
Dead |
Dead |
|
v50 |
0 |
0 |
0 |
0 |
0 |
0 |
Dead |
Dead |
|
PTCV |
0 |
11 |
896 |
576 |
1 152 |
384 |
448 |
640 |
|
vRVH |
0 |
22 |
154 |
269 |
378 |
106 |
198 |
538 |
|
vRVH+vRVF |
0 |
10 |
157 |
158 |
333 |
147 |
243 |
301 |
|
vRVF |
0 |
2 |
6 |
8 |
6 |
10 |
72 |
1 347 |
In the smallpox eradication campaign of the World Health Organization (WHO), vaccine was produced by extensively scarifying the skin of a calf and seeding the wounds with vaccinia virus. The scabs from the calf were collected after seven days, ground in saline, filtered, and the filtrate used for the vaccination of humans after testing for pyrogenicity. One calf yielded over 250 000 doses of vaccine. Once lyophilized, smallpox vaccine is very stable for several years. Smallpox vaccine was administered by scarifying the skin using a bifurcated needle or an airgun injector. Similar production and delivery procedures could be used for a vaccinia virus recombinant vaccine, or it could be propagated in tissue culture when facilities are available.
The prototype vaccinia virus recombinant vaccine is now being tested for potency, innocuity and safety and it is hoped that in future it may be of significant assistance for rinderpest control and eradication in developing countries.
Anderson, P.K., Fennie, H. & Yilma, T. 1988. Enhancement of a secondary antibody response by interferon-gamma treatment at primary immunization. J. Immunol., 40: 3599-3604.
Flexner, C., Hugin, A. & Moss, B. 1987. Prevention of vaccinia virus infection in immunodeficient mice by vector-directed IL-2 expression. Nature, 330: 259-262.
Grubman, M., Mebus, C., Dale, B., Yamanaka, M. & Yilma, T. 1988. Analysis of the polypeptides synthesized in rinderpest virus-infected cells. Virology, 163: 261 -267.
Hsu, D., Yamanaka, M., Miller, J., Dale, B., Grubman, M. & Yilma, T. 1988. Cloning of the fusion gene of rinderpest virus: comparative sequence analysis with other morbilliviruses. Virology, 166: 149-153.
Mackett, M., Yilma, T., Rose, J.K. & Moss, B. 1985. Vaccinia virus recombinants: expression of VSV genes and protective immunization of mice and cattle. Science, 227: 433-435.
Moss, B. 1974. Reproduction of poxviruses. In E. Frankel-Conrat, ed. Comprehensive Virology, vol. 3: 405-474. New York, Plenum Press.
Plowright, W. 1968. Virology monographs 3: rinderpest virus. p. 25-110. New York/Vienna, Springer-Verlag.
Yamanaka, M., Hsu, D., Dale, B., Crisp, T., Grubman, M. & Yilma, T. 1988. Cloning and sequence analysis of the haemagglutinin gene of the virulent strain of rinderpest virus. Virology, 166: 251-253.
Yilma, T., Anderson, K., Brechling, K. & Moss, B. 1987. In R.M. Chanock, H. Giensburg, R. Lerner & F. Brown, eds. Vaccines 87. Modern Approaches to New Vaccines including Prevention of AIDS. p. 393-396. Cold Spring Harbor. NY, Cold Spring Harbor Laboratory.
Yilma, T., Hsu, D., Jones, L., Owens, S., Grubman, M., Mebus, C., Yamanaka, M. & Dale, B. 1988. Protection of cattle against rinderpest with infectious vaccinia virus recombinants expressing the HA or F gene. Science, 242: 1058-1061.
