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Evaluation of a Live-Attenuated Foot-and-Mouth Disease Virus as a Vaccine Candidate
VIROLOGY
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Evaluation of a Live-Attenuated Foot-and-Mouth Disease Virus as a Vaccine Candidate P. W. MASON,* M. E. PICCONE,*,1 T. ST.-C. MCKENNA,† J. CHINSANGARAM,* and M. J. GRUBMAN*,2 *ARS, Plum Island Animal Disease Center and †Foreign Animal Disease Diagnostic Laboratory, U.S. Department of Agriculture, P. O. Box 848, Greenport, New York 11944 Received August 9, 1996; returned to author for revision September 10, 1996; accepted October 24, 1996 A variant of foot-and-mouth disease virus (FMDV) lacking the leader (L) coding region (A12-LLV2) was previously constructed and shown to be less virulent in cattle than its wild-type parent (A12-IC). In this study, cattle were tested for their clinical and immunological responses to subcutaneous inoculation with A12-LLV2 or A12-IC or to intramuscular vaccination with chemically inactivated A12-IC. Five weeks postinoculation animals were challenged by intradermal inoculation in the tongue with a virulent cattle-passaged virus. A12-LLV2-inoculated animals showed no clinical signs of disease and developed a neutralizing antibody response by 4 days postinoculation, whereas a companion control bovine did not seroconvert. After challenge, two of three inoculated animals did not develop lesions, but showed mild signs of infection. The third inoculated animal developed some lesions, but these were less severe than in the uninoculated control animal, which showed classical FMD. All animals inoculated with A12-IC developed a fever, two showed typical FMD lesions, and the companion control seroconverted, indicating that it had acquired infection by contact. The A12-IC-inoculated animals and the control were protected from challenge. Animals vaccinated with inactivated virus showed no clinical signs of disease and developed a neutralizing antibody response, and the control did not seroconvert. Upon challenge none of the vaccinated animals developed lesions, one developed a fever, and the control developed FMD. These experiments demonstrate the potential of a rationally designed live-attenuated FMDV vaccine. q 1997 Academic Press
INTRODUCTION
al., 1981; Beck and Strohmaier, 1987), the development of the carrier state in some vaccinated animals following contact with FMDV (Bachrach, 1968, 1978; Salt, 1993), and the relatively short-lived immunity of vaccinated animals, have been identified (Bachrach, 1968). Various workers have attempted to develop live-attenuated virus vaccines, derived by passage in alternate hosts and/or in tissue culture, but it has been difficult to obtain viruses that are both attenuated and immunogenic (Bachrach, 1968; Brooksby, 1982). Like other picornaviruses, FMDV contains a singlestranded, positive-sense RNA genome of approximately 8300 nucleotides surrounded by an icosahedral capsid containing 60 copies of each of 4 structural proteins. The RNA genome contains a single, long open reading frame that codes for the 4 structural proteins and a number of nonstructural proteins which function in various aspects of the virus replication cycle. FMDV and cardioviruses also contain a homopolymeric poly(C) tract of unknown function close to the 5* end of the genome (Brown et al., 1974). For FMDV it has been suggested that the poly(C) tract has a role in determining virulence (Harris and Brown, 1977), but other studies cast doubt on this possibility (Costa Giomi et al., 1988; Escarmis et al., 1992). However, genetically engineered mengoviruses containing short poly(C) tracts have been produced (Duke and Palmenberg, 1989) and shown to be dramatically attenuated in mice, swine, and other nonhuman primates
Foot-and-mouth disease (FMD), caused by a member of the Aphthovirus genus of the family Picornaviridae, is a highly contagious disease of cloven-hoofed animals that is spread by aerosol. The disease is characterized by temporary and debilitating oral and pedal vesicles which can result in a significant decline in production of meat or dairy products. Because of these problems and its highly infectious nature, trade embargos are enforced against countries with FMD, making it the most economically important disease of livestock worldwide (Bachrach, 1978; McCauley et al., 1979). Thus, most countries which are free of FMD maintain rigid quarantine and import restrictions to prevent its introduction, while control programs to eliminate the disease include slaughter of infected and exposed animals and ring vaccination using killed vaccine. The development of a chemically inactivated FMDV vaccine has led to the successful eradication of the disease from western Europe (Brown, 1992) and to its gradual elimination from other countries. Although these killed vaccines have been effective, recurrent problems, including shortcomings in vaccine manufacture (King et 1 Present address: Instituto de Biotecnologõ´a, CICV-INTA, C.C. 77 (1708) Moro´n, Buenos Aires, Argentina. 2 To whom correspondence and reprint requests should be addressed. Fax: (516) 323-2507. E-mail: [email protected].
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Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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(Osorio et al., 1996). In contrast, we demonstrated that genetically engineered FMDV with a poly(C) tract of 2 nucleotides is virulent in mice (Rieder et al., 1993) and likewise studies have shown that encephalomyocarditis viruses containing short poly(C) tracts are only slightly attenuated, if at all, compared to wild-type virus (Hahn and Palmenberg, 1995). Our current approach toward development of a liveattenuated FMDV is to manipulate the coding region of the genome. One of the viral nonstructural proteins, leader (L), is a papain-like proteinase (Gorbalenya et al., 1991; Kleina and Grubman, 1992; Piccone et al., 1995b; Roberts and Belsham, 1995) that autocatalytically cleaves itself from the polyprotein (Strebel and Beck, 1986) and cleaves p220 (recently named eIF-4G), a subunit of the cap-binding protein complex eIF-4F involved in the initiation of translation at the 5* end of most eukaryotic mRNAs (Devaney et al., 1988; Kirchweger et al., 1994; Medina et al., 1993). Cleavage of p220 correlates with the shutoff of cap-dependent host protein synthesis (Etchison et al., 1982) which occurs in FMDV-infected cells, allowing for unimpeded translation of viral RNA which is cap-independent. We have recently constructed a genetically altered variant of FMDV which is lacking the coding region for the L proteinase (Piccone et al., 1995a). The leaderless virus (A12-LLV2) replicates more slowly than wild-type (WT) virus in BHK-21 cells and is slightly attenuated in suckling mice (Piccone et al., 1995a). In a recent study in bovines, we showed that after aerosol exposure A12LLV2 is less virulent relative to parental WT virus derived from a full-length infectious clone (A12-IC), is less widely disseminated in the lung, and does not spread systemically to epithelial sites in the feet (Brown et al., 1996). The greatly reduced pathogenicity of A12-LLV2 in bovines identifies it as a candidate for a modified-live viral vaccine. In the present study, we have examined the virulence and immunogenicity of A12-LLV2 in bovines after subcutaneous inoculation and compared its potency and efficacy to a binary ethylenimine (BEI)-inactivated virus vaccine following challenge by intradermal inoculation in the tongue (IDL) with a virulent cattle-passaged A12 virus.
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of FMDV type A12 (Vallee strain 119, cattle passage 78) was used as the challenge virus (Cottral et al., 1965; McKenna et al., 1995). Bovines Twelve Hereford steers, aged 9–12 months, were used in the study. Animals were sorted into three test groups of four animals each and housed in separate rooms. In each group there were three inoculated animals and one uninoculated control. Each group of three animals was inoculated subcutaneously with either 107 PFU A12-LLV2 or A12-IC (corresponding to approximately 1 mg of virus) or intramuscularly (the standard route used in the field) with 2 mg of sucrose gradient-purified BEI-inactivated A12-IC (Bahnemann, 1990) emulsified in mineral oil (9:1 Marcol 52/Montanide 888). The animals were challenged by IDL inoculation at 35–36 days postinoculation with 104 PFU of the virulent cattle-passaged virus (approximately 20,000 cattle-infectious doses) and observed daily for clinical signs including fever and vesicular lesions. Serological assays Blood was collected at regular intervals pre- and postchallenge, and serum was separated, heat-inactivated at 567 for 30 min, stored at 0707, and subsequently assayed for neutralizing antibodies by a plaque reduction assay in BHK-21 cells. Neutralization titers were reported as the log of serum dilution yielding a 70% reduction in PFU (PRN70). Just prior to challenge, nasal secretions were obtained (Israel et al., 1992) and assayed for neutralizing antibodies by PRN70 . To determine if animals were virus carriers, esophagopharyngeal (OP) samples were collected, homogenized, treated with freon, filtered, and blind-passaged on BHK-21 cells for up to 3 days. Supernatants were titered and if plaques were obtained a PRN assay was performed with serum from a bovine that had been vaccinated with BEI-inactivated FMDV A12 to confirm that the virus was FMDV. RESULTS Experimental outline
Baby hamster kidney (BHK) cells (strain 21, clone 13) were used to propagate virus stocks and were used in plaque assays. Parental WT FMDV A12 119 (A12-IC) was derived from the full-length infectious clone pRMC35 (Rieder et al., 1993), and A12-LLV2 was derived from the infectious clone lacking the Lb coding region pRM-LLV2 (Piccone et al., 1995a). Fourth-passage virus stocks were used in all experiments. A virulent cattle-passaged strain
To compare the virulence of A12-IC with A12-LLV2 a preliminary experiment was performed by intradermal inoculation of bovines at multiple sites on the tongue with different amounts of each virus. As a control, a virulent cattle-passaged virus was also used and the animals were monitored clinically. This route of inoculation has been shown to be an extremely sensitive method of detecting the presence of FMDV (Henderson, 1952). The animal inoculated with the cattle-derived virus developed coalescing tongue lesions at the lowest dose used, 5 PFU, by 2 days postinoculation (dpi) and by 3 days lesions were present on the feet. The animal inoculated
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FIG. 1. Inoculation of bovines with A12-LLV2 and challenge with virulent virus. Three bovines, Nos. 13, 73, and 74, were inoculated subcutaneously with 107 PFU A12-LLV2 and a fourth animal, No. 9, housed in the same room, served as a control. The animals were observed daily for clinical signs including fever and lesions and were bled regularly, and serum and nasal secretions were tested by PRN70 . At 35 dpi the animals were challenged by IDL inoculation with 104 PFU of cattle-passaged A12 virus and observed as above.
with A12-IC showed coalescing lesions only after 3 days at 5 PFU. However, the A12-LLV2-inoculated animal did not have any lesions even at 725,000 PFU. This experiment showed that, in the bovine, A12-LLV2 was at least 105-fold less virulent than its parent, A12-IC, which in turn was slightly less virulent than the cattle-passaged virus. Based on this preliminary observation, a series of experiments was conducted to characterize the virulence of A12-LLV2 and A12-IC in greater detail and determine their potency and efficacy compared to the standard BEIinactivated vaccine. Animals given A12-LLV2 or A12-IC were inoculated subcutaneously, since we had previously demonstrated that this route induced a significant neutralizing antibody response in bovines (data not shown). Animals were vaccinated with BEI-inactivated virus by the approved standard intramuscular route. Three animals per group were inoculated once with 107 PFU A12-LLV2 or A12-IC, or 2 mg BEI-inactivated A12-IC. A control, noninoculated, animal was placed in the room with each of the above groups.
None of the inoculated animals, Nos. 13, 73, and 74, showed clinical signs (Fig. 1), but all developed a neu-
tralizing antibody response by 4 dpi (PRN70 from 1.6 to 2.5) which increased at 7 dpi (PRN70 from 2.3 to 3.2) and remained essentially constant until the animals were challenged at 35 dpi. The control animal, No. 9, did not seroconvert and showed no clinical signs of disease. At 35 dpi nasal secretions were obtained from each animal and only the inoculated animals showed a low-level neutralizing antibody response (Table 1). The four animals were challenged, at 35 dpi, by IDL inoculation with 104 PFU of a virulent cattle-passaged A12 virus (Fig. 1). The control animal, No. 9, developed a fever on the first day postchallenge and it remained over 39.57 for 4 consecutive days. This animal developed lesions on all four feet. Bovine No. 73, which had the highest neutralizing antibody response, developed a fever of over 39.57 for only 1 day and did not develop any pedal lesions. Bovine No. 74 developed a fever of over 39.57 for 4 days, but did not show any pedal lesions. Bovine No. 13, which had the lowest neutralizing antibody titer of the group, developed a fever for the first 3 days after challenge, again from 7 to 13 days postchallenge (dpc) and showed lesions on some of the feet, but their appearance was delayed and less severe compared to bovine No. 9, which developed lesions on all four feet. No virus was detected in OP fluids collected at 2, 4, 8, 15, and 31 dpi from these animals. OP samples
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TABLE 1 Summary of Immune Responses to Inoculation, and Challenge Prechallengea
Postchallengeb
Vaccinec
Animal No.
Serum NTd
Mucosal NTe
Serum NTd
Disease?f
None A12-LLV2 A12-LLV2 A12-LLV2
9 13 73 74
õ0.6 2.0 2.9 2.6
õ0.6 0.6 0.6 0.6
4.2 4.9 4.3 4.6
// / 0 {
Noneg A12-IC A12-IC A12-IC
56 40 47 58
3.3 3.6 3.6 3.9
1.5 1.8 ú2.1 1.2
3.5 3.3 3.3 3.6
0 0 0 0
None BEI-virus BEI-virus BEI-virus
50 51 52 55
0.6 3.8 2.9 4.1
õ0.9 3.2 1.8 2.9
4.5 4.5 4.8 (Died)h
// 0 { (Died)h
a
Serum and nasal secretions collected 35 or 36 days postinoculation. Serum samples collected 13–15 days postchallenge. c A12-LLV2 and A12-IC were administered subcutaneously; BEI-inactivated virus was given intramuscularly in oil. d Log of serum dilution giving a 70% reduction in PFU (see Materials and Methods). e Log of mucosal secretion dilution giving a 70% reduction in PFU (see Materials and Methods). f Disease profile: //, lesions on all four feet; /, lesions on one or more feet; {, no pedal lesions, but fever ú407 for more than 3 days; 0, no pedal lesions or fever of ú407 for more than 1 day. g Animal apparently acquired infection from jointly housed A12-IC inoculated animals. h Animal No. 55 died Day 4 postchallenge from non-FMD causes; see text. b
were collected at various times from 30 to 108 dpc and virus was isolated from bovine No. 9 at 40 and 46 dpc, while of the inoculated animals, only No. 73 had virus and only at 44 dpc. A12-IC-inoculated animals Animals inoculated with the parental WT virus, Nos. 40, 47, and 58, developed typical signs of FMD, i.e., fever and epithelial lesions, and all three animals had high levels of neutralizing antibody (Fig. 2). The control animal, No. 56, seroconverted by 15 dpi (PRN70 3.8), but showed no signs of FMD. All four animals developed a relatively high neutralizing antibody titer in their nasal secretions (Table 1). Upon challenge all four animals were fully protected and showed no increase in neutralizing antibody titer. Virus was recovered in the OP samples of the control animal at 8 and 30 dpi and in No. 47 at 15 dpi, but was not recovered from any of the other samples collected up to 76 dpc.
oped typical signs of FMD and seroconverted. Of the three vaccinated animals, No. 55, which had the highest prechallenge neutralizing antibody titer, died on the third day after challenge from non-FMD causes. Bovine No. 52, which had the lowest prechallenge titer, had a fever of over 407 for 3 days and an increase in neutralizing antibody titer, but no other signs of FMD. Bovine No. 51 was completely protected from disease, but had a slight rise in neutralizing antibody titer. No virus was detected in OP fluids collected at 32, 34, 67, or 74 dpc. DISCUSSION
The three vaccinated animals, Nos. 51, 52, and 55, showed no signs of disease, developed significant serum neutralizing antibody titers as well as high neutralizing titers in nasal secretions (Fig. 3 and Table 1). The control animal, No. 50, showed no signs of disease and did not seroconvert. Upon challenge the control animal devel-
In this communication, we have demonstrated that a genetically altered variant of FMDV, A12-LLV2, can be safely administered to cattle and that it induces a neutralizing and protective antibody response. The inocuity of A12-LLV2 was shown by direct inoculation into the tongue, supporting our recent studies showing that aerosol exposure of bovines to A12-LLV2 failed to produce disease (Brown et al., 1996). Additionally, in contrast to WT virus, A12-LLV2 did not spread from the subcutaneously inoculated animals to an uninoculated control. These experiments demonstrate that 107 to 108 PFU A12LLV2 is nonpathogenic in bovines, yet one dose of the virus is able to induce a rapid immune response. Upon challenge by IDL inoculation with approximately 20,000 cattle-infectious doses of a virulent cattle-passaged vi-
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FIG. 2. Inoculation of bovines with A12-IC and challenge with virulent virus. Three bovines, Nos. 40, 47, and 58, were inoculated subcutaneously with 107 PFU A12-IC and a fourth animal, No. 56, housed in the same room, served as a control. The animals were observed daily for clinical signs including fever and lesions and were bled regularly, and serum and nasal secretions were tested by PRN70 . At 36 dpi the animals were challenged by IDL inoculation with 104 PFU of cattle-passaged A12 virus and observed as above.
rus, two of three A12-LLV2-inoculated animals were protected from disease (pedal vesicles) and the third animal had a disease of reduced severity. In this study, the level of protection of cattle following A12-LLV2 inoculation was similar to the protection afforded cattle after vaccination with standard BEI-inactivated virus. It should be emphasized that the route of challenge used in this study is severe, since virus is directly inoculated into susceptible tissue where it is able to multiply sufficiently to produce a lesion. In previous studies, cattle vaccinated with a similar BEI-inactivated virus were fully protected when challenged by contact exposure to an infected pig (McKenna et al., 1995). The potency test we utilized, IDL inoculation with 20,000 cattle-infectious doses on the upper surface of the tongue followed by scoring transmission as the presence of lesions on the feet, is similar to that recommended by the Office International des Epizooties (Donaldson and Doel, 1992). Historically, attenuated FMDV vaccines have been produced by passage in alternate hosts and/or in tissue culture (Brooksby, 1982). However, it proved difficult to develop strains that were both attenuated and able to induce an immunogenic response in cattle (Brooksby, 1982). An additional problem with live-attenuated viruses is the possibility of reversion to virulence. Over the past
15 years, we have acquired comprehensive information on the FMDV genome, including the identification and partial characterization of all the viral-encoded structural and nonstructural proteins and knowledge of their role in virus replication, as well as an understanding of the function of noncoding regions of the viral genome. More recently the complete nucleotide sequence of a number of subtypes and serotypes of FMDV has been determined and infectious full-length cDNA clones of type O1 (Zibert et al., 1990) and A12 (Rieder et al., 1993) have been constructed. This extensive knowledge of the virus at the molecular level and the relative ease of manipulation of recombinant DNA allows for the rational development of a live-attenuated viral vaccine from full-length cDNA clones. Based on this information, we have constructed a genetic variant of FMDV that has a complete deletion of a viral coding region. This approach, as opposed to passage on unnatural hosts which selects point mutations, significantly reduces the risk of this virus reverting to virulence. The leader coding region of FMDV is at the 5* end of the long open reading frame and codes for a proteinase that is directly involved in the shutoff of host protein synthesis as a result of the cleavage of a translation initiation factor required for the cap-dependent transla-
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FIG. 3. Vaccination of bovines with BEI-inactivated A12-IC and challenge with virulent virus. Three bovines, Nos. 51, 52, and 55, were inoculated intramuscularly with 2 mg BEI-inactivated A12-IC and a fourth animal, No. 50, housed in the same room, served as a control. The animals were observed daily for clinical signs including fever and lesions and were bled regularly, and serum and nasal secretions were tested by PRN70 . At 35 dpi the animals were challenged by IDL inoculation with 104 PFU of cattle-passaged A12 virus and observed as above. *Died day 4 postchallenge.
tion of most eukaryotic mRNAs (Devaney et al., 1988; Medina et al., 1993; Kirchweger et al., 1994). Deletion of this coding region should result in a virus that replicates more slowly than WT virus, since it has lost a portion of its competitive advantage over cellular mRNAs. Initial experiments in tissue culture and animals support this expectation. In BHK-21 cells, we have observed that A12LLV2 replicates more slowly than WT virus in a singlecycle growth curve, is less efficient in host cell protein synthesis shutoff, and is delayed in the initiation of viral protein synthesis (Piccone et al., 1995a). Our recent studies on the pathogenesis of FMD in cattle after aerosol exposure to either A12-LLV2 or A12-IC indicated that after initial infection of the respiratory bronchioles there was little dissemination in the lungs and no detectable virus at secondary sites following A12-LLV2 infection in contrast to A12-IC infection (Brown et al., 1996). Furthermore, in preliminary experiments with two primary cell lines derived from susceptible animals, WT virus grows rapidly and to high titers, while A12-LLV2 replicates slowly and is unable to spread to neighboring cells (unpublished observations). Studies examining the host response to A12-LLV2 infection may help us understand the apparent inability of this virus to spread and shed light on host mechanisms which may be relevant to control of FMD.
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The results of our experiments with A12-LLV2 suggest that a leaderless virus could be used as a possible FMD vaccine. Additional studies identifying the optimal regimen of live-attenuated virus inoculation are planned. The possibility that intranasal inoculation, which would mimic the natural route of infection, could induce local mucosal immunity as an additional barrier against virus replication is worth examining. ACKNOWLEDGMENTS We thank Marla Zellner for expert technical assistance and the Plum Island Animal Disease Center animal care staff for attention to the experimental animals.
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Evaluation of a Live-Attenuated Foot-and-Mouth Disease Virus as a Vaccine Candidate P. W. MASON,* M. E. PICCONE,*,1 T. ST.-C. MCKENNA,† J. CHINSANGARAM,* and M. J. GRUBMAN*,2 *ARS, Plum Island Animal Disease Center and †Foreign Animal Disease Diagnostic Laboratory, U.S. Department of Agriculture, P. O. Box 848, Greenport, New York 11944 Received August 9, 1996; returned to author for revision September 10, 1996; accepted October 24, 1996 A variant of foot-and-mouth disease virus (FMDV) lacking the leader (L) coding region (A12-LLV2) was previously constructed and shown to be less virulent in cattle than its wild-type parent (A12-IC). In this study, cattle were tested for their clinical and immunological responses to subcutaneous inoculation with A12-LLV2 or A12-IC or to intramuscular vaccination with chemically inactivated A12-IC. Five weeks postinoculation animals were challenged by intradermal inoculation in the tongue with a virulent cattle-passaged virus. A12-LLV2-inoculated animals showed no clinical signs of disease and developed a neutralizing antibody response by 4 days postinoculation, whereas a companion control bovine did not seroconvert. After challenge, two of three inoculated animals did not develop lesions, but showed mild signs of infection. The third inoculated animal developed some lesions, but these were less severe than in the uninoculated control animal, which showed classical FMD. All animals inoculated with A12-IC developed a fever, two showed typical FMD lesions, and the companion control seroconverted, indicating that it had acquired infection by contact. The A12-IC-inoculated animals and the control were protected from challenge. Animals vaccinated with inactivated virus showed no clinical signs of disease and developed a neutralizing antibody response, and the control did not seroconvert. Upon challenge none of the vaccinated animals developed lesions, one developed a fever, and the control developed FMD. These experiments demonstrate the potential of a rationally designed live-attenuated FMDV vaccine. q 1997 Academic Press
INTRODUCTION
al., 1981; Beck and Strohmaier, 1987), the development of the carrier state in some vaccinated animals following contact with FMDV (Bachrach, 1968, 1978; Salt, 1993), and the relatively short-lived immunity of vaccinated animals, have been identified (Bachrach, 1968). Various workers have attempted to develop live-attenuated virus vaccines, derived by passage in alternate hosts and/or in tissue culture, but it has been difficult to obtain viruses that are both attenuated and immunogenic (Bachrach, 1968; Brooksby, 1982). Like other picornaviruses, FMDV contains a singlestranded, positive-sense RNA genome of approximately 8300 nucleotides surrounded by an icosahedral capsid containing 60 copies of each of 4 structural proteins. The RNA genome contains a single, long open reading frame that codes for the 4 structural proteins and a number of nonstructural proteins which function in various aspects of the virus replication cycle. FMDV and cardioviruses also contain a homopolymeric poly(C) tract of unknown function close to the 5* end of the genome (Brown et al., 1974). For FMDV it has been suggested that the poly(C) tract has a role in determining virulence (Harris and Brown, 1977), but other studies cast doubt on this possibility (Costa Giomi et al., 1988; Escarmis et al., 1992). However, genetically engineered mengoviruses containing short poly(C) tracts have been produced (Duke and Palmenberg, 1989) and shown to be dramatically attenuated in mice, swine, and other nonhuman primates
Foot-and-mouth disease (FMD), caused by a member of the Aphthovirus genus of the family Picornaviridae, is a highly contagious disease of cloven-hoofed animals that is spread by aerosol. The disease is characterized by temporary and debilitating oral and pedal vesicles which can result in a significant decline in production of meat or dairy products. Because of these problems and its highly infectious nature, trade embargos are enforced against countries with FMD, making it the most economically important disease of livestock worldwide (Bachrach, 1978; McCauley et al., 1979). Thus, most countries which are free of FMD maintain rigid quarantine and import restrictions to prevent its introduction, while control programs to eliminate the disease include slaughter of infected and exposed animals and ring vaccination using killed vaccine. The development of a chemically inactivated FMDV vaccine has led to the successful eradication of the disease from western Europe (Brown, 1992) and to its gradual elimination from other countries. Although these killed vaccines have been effective, recurrent problems, including shortcomings in vaccine manufacture (King et 1 Present address: Instituto de Biotecnologõ´a, CICV-INTA, C.C. 77 (1708) Moro´n, Buenos Aires, Argentina. 2 To whom correspondence and reprint requests should be addressed. Fax: (516) 323-2507. E-mail: [email protected].
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(Osorio et al., 1996). In contrast, we demonstrated that genetically engineered FMDV with a poly(C) tract of 2 nucleotides is virulent in mice (Rieder et al., 1993) and likewise studies have shown that encephalomyocarditis viruses containing short poly(C) tracts are only slightly attenuated, if at all, compared to wild-type virus (Hahn and Palmenberg, 1995). Our current approach toward development of a liveattenuated FMDV is to manipulate the coding region of the genome. One of the viral nonstructural proteins, leader (L), is a papain-like proteinase (Gorbalenya et al., 1991; Kleina and Grubman, 1992; Piccone et al., 1995b; Roberts and Belsham, 1995) that autocatalytically cleaves itself from the polyprotein (Strebel and Beck, 1986) and cleaves p220 (recently named eIF-4G), a subunit of the cap-binding protein complex eIF-4F involved in the initiation of translation at the 5* end of most eukaryotic mRNAs (Devaney et al., 1988; Kirchweger et al., 1994; Medina et al., 1993). Cleavage of p220 correlates with the shutoff of cap-dependent host protein synthesis (Etchison et al., 1982) which occurs in FMDV-infected cells, allowing for unimpeded translation of viral RNA which is cap-independent. We have recently constructed a genetically altered variant of FMDV which is lacking the coding region for the L proteinase (Piccone et al., 1995a). The leaderless virus (A12-LLV2) replicates more slowly than wild-type (WT) virus in BHK-21 cells and is slightly attenuated in suckling mice (Piccone et al., 1995a). In a recent study in bovines, we showed that after aerosol exposure A12LLV2 is less virulent relative to parental WT virus derived from a full-length infectious clone (A12-IC), is less widely disseminated in the lung, and does not spread systemically to epithelial sites in the feet (Brown et al., 1996). The greatly reduced pathogenicity of A12-LLV2 in bovines identifies it as a candidate for a modified-live viral vaccine. In the present study, we have examined the virulence and immunogenicity of A12-LLV2 in bovines after subcutaneous inoculation and compared its potency and efficacy to a binary ethylenimine (BEI)-inactivated virus vaccine following challenge by intradermal inoculation in the tongue (IDL) with a virulent cattle-passaged A12 virus.
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of FMDV type A12 (Vallee strain 119, cattle passage 78) was used as the challenge virus (Cottral et al., 1965; McKenna et al., 1995). Bovines Twelve Hereford steers, aged 9–12 months, were used in the study. Animals were sorted into three test groups of four animals each and housed in separate rooms. In each group there were three inoculated animals and one uninoculated control. Each group of three animals was inoculated subcutaneously with either 107 PFU A12-LLV2 or A12-IC (corresponding to approximately 1 mg of virus) or intramuscularly (the standard route used in the field) with 2 mg of sucrose gradient-purified BEI-inactivated A12-IC (Bahnemann, 1990) emulsified in mineral oil (9:1 Marcol 52/Montanide 888). The animals were challenged by IDL inoculation at 35–36 days postinoculation with 104 PFU of the virulent cattle-passaged virus (approximately 20,000 cattle-infectious doses) and observed daily for clinical signs including fever and vesicular lesions. Serological assays Blood was collected at regular intervals pre- and postchallenge, and serum was separated, heat-inactivated at 567 for 30 min, stored at 0707, and subsequently assayed for neutralizing antibodies by a plaque reduction assay in BHK-21 cells. Neutralization titers were reported as the log of serum dilution yielding a 70% reduction in PFU (PRN70). Just prior to challenge, nasal secretions were obtained (Israel et al., 1992) and assayed for neutralizing antibodies by PRN70 . To determine if animals were virus carriers, esophagopharyngeal (OP) samples were collected, homogenized, treated with freon, filtered, and blind-passaged on BHK-21 cells for up to 3 days. Supernatants were titered and if plaques were obtained a PRN assay was performed with serum from a bovine that had been vaccinated with BEI-inactivated FMDV A12 to confirm that the virus was FMDV. RESULTS Experimental outline
Baby hamster kidney (BHK) cells (strain 21, clone 13) were used to propagate virus stocks and were used in plaque assays. Parental WT FMDV A12 119 (A12-IC) was derived from the full-length infectious clone pRMC35 (Rieder et al., 1993), and A12-LLV2 was derived from the infectious clone lacking the Lb coding region pRM-LLV2 (Piccone et al., 1995a). Fourth-passage virus stocks were used in all experiments. A virulent cattle-passaged strain
To compare the virulence of A12-IC with A12-LLV2 a preliminary experiment was performed by intradermal inoculation of bovines at multiple sites on the tongue with different amounts of each virus. As a control, a virulent cattle-passaged virus was also used and the animals were monitored clinically. This route of inoculation has been shown to be an extremely sensitive method of detecting the presence of FMDV (Henderson, 1952). The animal inoculated with the cattle-derived virus developed coalescing tongue lesions at the lowest dose used, 5 PFU, by 2 days postinoculation (dpi) and by 3 days lesions were present on the feet. The animal inoculated
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FIG. 1. Inoculation of bovines with A12-LLV2 and challenge with virulent virus. Three bovines, Nos. 13, 73, and 74, were inoculated subcutaneously with 107 PFU A12-LLV2 and a fourth animal, No. 9, housed in the same room, served as a control. The animals were observed daily for clinical signs including fever and lesions and were bled regularly, and serum and nasal secretions were tested by PRN70 . At 35 dpi the animals were challenged by IDL inoculation with 104 PFU of cattle-passaged A12 virus and observed as above.
with A12-IC showed coalescing lesions only after 3 days at 5 PFU. However, the A12-LLV2-inoculated animal did not have any lesions even at 725,000 PFU. This experiment showed that, in the bovine, A12-LLV2 was at least 105-fold less virulent than its parent, A12-IC, which in turn was slightly less virulent than the cattle-passaged virus. Based on this preliminary observation, a series of experiments was conducted to characterize the virulence of A12-LLV2 and A12-IC in greater detail and determine their potency and efficacy compared to the standard BEIinactivated vaccine. Animals given A12-LLV2 or A12-IC were inoculated subcutaneously, since we had previously demonstrated that this route induced a significant neutralizing antibody response in bovines (data not shown). Animals were vaccinated with BEI-inactivated virus by the approved standard intramuscular route. Three animals per group were inoculated once with 107 PFU A12-LLV2 or A12-IC, or 2 mg BEI-inactivated A12-IC. A control, noninoculated, animal was placed in the room with each of the above groups.
None of the inoculated animals, Nos. 13, 73, and 74, showed clinical signs (Fig. 1), but all developed a neu-
tralizing antibody response by 4 dpi (PRN70 from 1.6 to 2.5) which increased at 7 dpi (PRN70 from 2.3 to 3.2) and remained essentially constant until the animals were challenged at 35 dpi. The control animal, No. 9, did not seroconvert and showed no clinical signs of disease. At 35 dpi nasal secretions were obtained from each animal and only the inoculated animals showed a low-level neutralizing antibody response (Table 1). The four animals were challenged, at 35 dpi, by IDL inoculation with 104 PFU of a virulent cattle-passaged A12 virus (Fig. 1). The control animal, No. 9, developed a fever on the first day postchallenge and it remained over 39.57 for 4 consecutive days. This animal developed lesions on all four feet. Bovine No. 73, which had the highest neutralizing antibody response, developed a fever of over 39.57 for only 1 day and did not develop any pedal lesions. Bovine No. 74 developed a fever of over 39.57 for 4 days, but did not show any pedal lesions. Bovine No. 13, which had the lowest neutralizing antibody titer of the group, developed a fever for the first 3 days after challenge, again from 7 to 13 days postchallenge (dpc) and showed lesions on some of the feet, but their appearance was delayed and less severe compared to bovine No. 9, which developed lesions on all four feet. No virus was detected in OP fluids collected at 2, 4, 8, 15, and 31 dpi from these animals. OP samples
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TABLE 1 Summary of Immune Responses to Inoculation, and Challenge Prechallengea
Postchallengeb
Vaccinec
Animal No.
Serum NTd
Mucosal NTe
Serum NTd
Disease?f
None A12-LLV2 A12-LLV2 A12-LLV2
9 13 73 74
õ0.6 2.0 2.9 2.6
õ0.6 0.6 0.6 0.6
4.2 4.9 4.3 4.6
// / 0 {
Noneg A12-IC A12-IC A12-IC
56 40 47 58
3.3 3.6 3.6 3.9
1.5 1.8 ú2.1 1.2
3.5 3.3 3.3 3.6
0 0 0 0
None BEI-virus BEI-virus BEI-virus
50 51 52 55
0.6 3.8 2.9 4.1
õ0.9 3.2 1.8 2.9
4.5 4.5 4.8 (Died)h
// 0 { (Died)h
a
Serum and nasal secretions collected 35 or 36 days postinoculation. Serum samples collected 13–15 days postchallenge. c A12-LLV2 and A12-IC were administered subcutaneously; BEI-inactivated virus was given intramuscularly in oil. d Log of serum dilution giving a 70% reduction in PFU (see Materials and Methods). e Log of mucosal secretion dilution giving a 70% reduction in PFU (see Materials and Methods). f Disease profile: //, lesions on all four feet; /, lesions on one or more feet; {, no pedal lesions, but fever ú407 for more than 3 days; 0, no pedal lesions or fever of ú407 for more than 1 day. g Animal apparently acquired infection from jointly housed A12-IC inoculated animals. h Animal No. 55 died Day 4 postchallenge from non-FMD causes; see text. b
were collected at various times from 30 to 108 dpc and virus was isolated from bovine No. 9 at 40 and 46 dpc, while of the inoculated animals, only No. 73 had virus and only at 44 dpc. A12-IC-inoculated animals Animals inoculated with the parental WT virus, Nos. 40, 47, and 58, developed typical signs of FMD, i.e., fever and epithelial lesions, and all three animals had high levels of neutralizing antibody (Fig. 2). The control animal, No. 56, seroconverted by 15 dpi (PRN70 3.8), but showed no signs of FMD. All four animals developed a relatively high neutralizing antibody titer in their nasal secretions (Table 1). Upon challenge all four animals were fully protected and showed no increase in neutralizing antibody titer. Virus was recovered in the OP samples of the control animal at 8 and 30 dpi and in No. 47 at 15 dpi, but was not recovered from any of the other samples collected up to 76 dpc.
oped typical signs of FMD and seroconverted. Of the three vaccinated animals, No. 55, which had the highest prechallenge neutralizing antibody titer, died on the third day after challenge from non-FMD causes. Bovine No. 52, which had the lowest prechallenge titer, had a fever of over 407 for 3 days and an increase in neutralizing antibody titer, but no other signs of FMD. Bovine No. 51 was completely protected from disease, but had a slight rise in neutralizing antibody titer. No virus was detected in OP fluids collected at 32, 34, 67, or 74 dpc. DISCUSSION
The three vaccinated animals, Nos. 51, 52, and 55, showed no signs of disease, developed significant serum neutralizing antibody titers as well as high neutralizing titers in nasal secretions (Fig. 3 and Table 1). The control animal, No. 50, showed no signs of disease and did not seroconvert. Upon challenge the control animal devel-
In this communication, we have demonstrated that a genetically altered variant of FMDV, A12-LLV2, can be safely administered to cattle and that it induces a neutralizing and protective antibody response. The inocuity of A12-LLV2 was shown by direct inoculation into the tongue, supporting our recent studies showing that aerosol exposure of bovines to A12-LLV2 failed to produce disease (Brown et al., 1996). Additionally, in contrast to WT virus, A12-LLV2 did not spread from the subcutaneously inoculated animals to an uninoculated control. These experiments demonstrate that 107 to 108 PFU A12LLV2 is nonpathogenic in bovines, yet one dose of the virus is able to induce a rapid immune response. Upon challenge by IDL inoculation with approximately 20,000 cattle-infectious doses of a virulent cattle-passaged vi-
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FIG. 2. Inoculation of bovines with A12-IC and challenge with virulent virus. Three bovines, Nos. 40, 47, and 58, were inoculated subcutaneously with 107 PFU A12-IC and a fourth animal, No. 56, housed in the same room, served as a control. The animals were observed daily for clinical signs including fever and lesions and were bled regularly, and serum and nasal secretions were tested by PRN70 . At 36 dpi the animals were challenged by IDL inoculation with 104 PFU of cattle-passaged A12 virus and observed as above.
rus, two of three A12-LLV2-inoculated animals were protected from disease (pedal vesicles) and the third animal had a disease of reduced severity. In this study, the level of protection of cattle following A12-LLV2 inoculation was similar to the protection afforded cattle after vaccination with standard BEI-inactivated virus. It should be emphasized that the route of challenge used in this study is severe, since virus is directly inoculated into susceptible tissue where it is able to multiply sufficiently to produce a lesion. In previous studies, cattle vaccinated with a similar BEI-inactivated virus were fully protected when challenged by contact exposure to an infected pig (McKenna et al., 1995). The potency test we utilized, IDL inoculation with 20,000 cattle-infectious doses on the upper surface of the tongue followed by scoring transmission as the presence of lesions on the feet, is similar to that recommended by the Office International des Epizooties (Donaldson and Doel, 1992). Historically, attenuated FMDV vaccines have been produced by passage in alternate hosts and/or in tissue culture (Brooksby, 1982). However, it proved difficult to develop strains that were both attenuated and able to induce an immunogenic response in cattle (Brooksby, 1982). An additional problem with live-attenuated viruses is the possibility of reversion to virulence. Over the past
15 years, we have acquired comprehensive information on the FMDV genome, including the identification and partial characterization of all the viral-encoded structural and nonstructural proteins and knowledge of their role in virus replication, as well as an understanding of the function of noncoding regions of the viral genome. More recently the complete nucleotide sequence of a number of subtypes and serotypes of FMDV has been determined and infectious full-length cDNA clones of type O1 (Zibert et al., 1990) and A12 (Rieder et al., 1993) have been constructed. This extensive knowledge of the virus at the molecular level and the relative ease of manipulation of recombinant DNA allows for the rational development of a live-attenuated viral vaccine from full-length cDNA clones. Based on this information, we have constructed a genetic variant of FMDV that has a complete deletion of a viral coding region. This approach, as opposed to passage on unnatural hosts which selects point mutations, significantly reduces the risk of this virus reverting to virulence. The leader coding region of FMDV is at the 5* end of the long open reading frame and codes for a proteinase that is directly involved in the shutoff of host protein synthesis as a result of the cleavage of a translation initiation factor required for the cap-dependent transla-
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FIG. 3. Vaccination of bovines with BEI-inactivated A12-IC and challenge with virulent virus. Three bovines, Nos. 51, 52, and 55, were inoculated intramuscularly with 2 mg BEI-inactivated A12-IC and a fourth animal, No. 50, housed in the same room, served as a control. The animals were observed daily for clinical signs including fever and lesions and were bled regularly, and serum and nasal secretions were tested by PRN70 . At 35 dpi the animals were challenged by IDL inoculation with 104 PFU of cattle-passaged A12 virus and observed as above. *Died day 4 postchallenge.
tion of most eukaryotic mRNAs (Devaney et al., 1988; Medina et al., 1993; Kirchweger et al., 1994). Deletion of this coding region should result in a virus that replicates more slowly than WT virus, since it has lost a portion of its competitive advantage over cellular mRNAs. Initial experiments in tissue culture and animals support this expectation. In BHK-21 cells, we have observed that A12LLV2 replicates more slowly than WT virus in a singlecycle growth curve, is less efficient in host cell protein synthesis shutoff, and is delayed in the initiation of viral protein synthesis (Piccone et al., 1995a). Our recent studies on the pathogenesis of FMD in cattle after aerosol exposure to either A12-LLV2 or A12-IC indicated that after initial infection of the respiratory bronchioles there was little dissemination in the lungs and no detectable virus at secondary sites following A12-LLV2 infection in contrast to A12-IC infection (Brown et al., 1996). Furthermore, in preliminary experiments with two primary cell lines derived from susceptible animals, WT virus grows rapidly and to high titers, while A12-LLV2 replicates slowly and is unable to spread to neighboring cells (unpublished observations). Studies examining the host response to A12-LLV2 infection may help us understand the apparent inability of this virus to spread and shed light on host mechanisms which may be relevant to control of FMD.
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The results of our experiments with A12-LLV2 suggest that a leaderless virus could be used as a possible FMD vaccine. Additional studies identifying the optimal regimen of live-attenuated virus inoculation are planned. The possibility that intranasal inoculation, which would mimic the natural route of infection, could induce local mucosal immunity as an additional barrier against virus replication is worth examining. ACKNOWLEDGMENTS We thank Marla Zellner for expert technical assistance and the Plum Island Animal Disease Center animal care staff for attention to the experimental animals.
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