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DI Particles: Painting a Clearer Picture of Viruses and Vaccines
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DI Particles: Painting a Clearer Picture of Viruses and Vaccines
T
he neat picture of the classic viral infection-virus entering the host cell, the taking over of the cell's machinery to manufacture uniform progeny virus which, in turn, enter other cells to continue the process until halted by the host's developing antibody to the virus-is undergoing some major rethinking. Hand-in-hand with this changing concept are emerging some novel ideas regarding the way virus replication occurs at the molecular level, the nature and control of viral infections, and the way in which vaccines work to protect against viral diseases. I This is the outcome of current research into viral infections and, in particular, into the formation of what are known as defective interfering particles-fragments of the virus that arise spontaneously as viruses infect and reproduce within the host cell. Defective interfering particles, or 01 particles as they are called, have been found in almost every virus in which they have been sought, according to Dr. Robert A. Lazzarini, acting chief of the laboratory of molecular genetics at the National Institute of Neurological and Communicative Disorders and Stroke at the National Institutes of Health. Although these particles contain all the viral proteins, they contain only a small piece of the viral chromosome-often less than one gene-so they can't carry out all the functions of the complete virus. They are much smaller than the complete parent virus. But in all their morphological features, they are exactly like the whole virus. And, despite their defects, when a cell in which they are present is infected with the parent virus, they can be replicated," Lazzarini observes. When DI particles are replicated, it is done at the expense of the fully II
14
infective parent virus. Thus their presence in a cell reduces the number of whole infective virus particles assembled in the cell. In the case of vesicular stomatitis virus (VSV), for example, a cell that is infected with a single virus would produce about 10,000 progeny particles. But if DI particles are in that cell as well, it may yield only 10 or 100 particles of the complete virus. Thus, DI particles are not only defective-they also exert an interfering effect on virus infection. "In fact," Lazzarini points out, "if you have enough 01 particles in an infected cell, you can snuff out the virus infection completely in that cell. We have also found that the 01 particles produced in these cells will penetrate and similarly protect other cells in the face of a similar infection ." Lazzarini and his staff do most of their work with VSV because it is handled easily and obtainable in large quantities. It is similar in shape to rabies virus, looking a little like a bacillus, sort of bullet-shaped. 01 particles are extremely specific, Lazzarini told American Pharmacy. A 01 particle from VSV will only inhibit replication of VSV; it won't inhibit any other type of virus.
Relatively Benign. Most 01 particles don't appear to have any effect on the host cell. "If you take a cell and add 1,000 or 10,000 DI particles nothing happens to the host cell that we can see," Lazzarini said. "The cell still goes on multiplying in the normal way. There are some exceptions; some 01 particles will kill the cell, but they are rare." In about 1970, Drs. Alice Huang and David Baltimore speculated that the inhibiting effect of 01 particles on cell replication played a role in the control of natural infections-in fact terminating them. The particles,
they held, acted as natural, virusspecific antiviral agents. They also speculated that 01 particles might be responsible for chronic virus infections by interfering with the replication of the full, infectious virus. This prevented what would otherwise have been an acute infection, they reasoned . "We know something about the origin of 01 particles," Lazzarini said. "In essence they arise because of a mistake in replication. As the parent virus is replicated in the cell and its genome is being copied, the enzyme (replicase) that copies the genome makes a mistake. But it is a curious kind of mistake. While a substantial amount of information is left out, the essential information for copying remains. You can't just take a fragment of a virus chromosome and expect it to be copied; the enzyme doesn't know where to start because it doesn't have the proper ends. But a DI particle does, and so it is copied. "If one is looking to make use of such information in therapy or prevention of viral diseases, a reasonable approach would be to find I chemicals that might stimulate such mistakes in replication," Lazzarini continued. "If these can be found, then we have a way of creating the antiviral component in situ; right in the very cells that are infected with the virus. These chemicals would be very specific and not likely to inter- I fere with any host cell functions because the host has a completely different kind of replication machinery than do most viruses. There is a certain aesthetic appeal in targeting antiviral therapy in the specific cell i tha t is infected." Lazzarini has not pursued this approach. . "We have rather limited resources here," he says, and we're mostly interested in the basic scientific as' pects." II
American Pharmacy Vol. NS21 , No. 10, October 1981/5
Possible Vaccines However, he does speculate free-
ly about ways in which OI particles could be used to control viral infections: purified populations of OI particles as a type of vaccine for instance. "The particles aren't infectious, they carry a full variety of all the virus proteins with them, they penetrate cells, and would stimulate the immune system. There's no reason to believe they aren't immunogenic," he says. Right now Lazzarini and his asso-
useful in making a vaccine." Lazzarini outlines one possibility: "Suppose it were possible to make a hybrid virus out of a fully attenuated vaccine strain by inserting a gene of a second virus into its chromosome. If the inserted piece were positioned properly so that it were expressed as protein, the hybrid vaccine virus might stimulate an immune response to both parent viruses. "Great care would have to be taken to ensure that the hybrid virus did not acquire any detrimental biological properties. Using this strat-
Drs. Lazzarini and Manfried Schubert discuss an autoradiogram of eiectrophoretically separated radioactive 01 particle nucleic acids.
ciates are looking into gene splicing techniques as an avenue that may be useful not only in producing a vaccine, but may throw light on the gene sequences that are of importance in OI particles-possibly of viruses as well. With the aid of recombinant DNA procedures they are building OI particles by copying the appropriate sequences. "Once they're in DNA, they can be easily manipulated and we can glue them together, rearrange them and so forth," he says. "We're interested in this approach because it enables us to demonstrate what sequences are important and what are not. On a possibly more practical level, we might be able to splice into a virus chromosome information that would, if it were expressed, be ~~e" [nCan Pharmacy Vol.
NS21 , No. 10, October 1981 /567
egy, it may be possible not only to produce multipurpose vaccines but to produce vaccines against viruses not easily 'tamed' or attenuated." It is also from Lazzarini's laboratory that some provocative findings are emanating on the way vaccines work. Studies with viruses that have been attenuated in the laboratory by adapting them to growth in chick embryo cells-a standard method of manufacturing vaccines-is throwing light on the role of OI particles in the way a vaccine confers immunity. One of Lazarrini's associates, Dr. Micheline McCarthy, working with a chick embryo adapted strain of mumps virus, has shown that, while this virus can infect mammalian cells, the infection is inapparent. The cells appear microscop-
ically normal. But on close study, one sees that there is mumps virus being replicated in these cells and that this infection is completely dominated by OI particles. "Now," says Lazzarini, "if you take this attenuated strain of virus and put it into chick embryo cells, then there is no evidence of OI particles, but one gets a fulminating, full-blown viral infection. Again, if one takes other strains of mumps virus that have not been adapted to chick embryo cells and puts these into mammalian cell cultures then one again sees a fulminating infection and no evidence of the presence of 01 particles. "The point is that where there are 01 particles present, there seems to be attenuation. It may be that attenuation by OI particles is common among vaccine strains," Lazzarin i maintains . "It may be that what on e wants in a vaccine is a virus that only gives a subclinical infection so that it doesn't cause disease, but also permits viral replication to go forward so that the immune response is kept in a constant state of stimulation. "That's one way of looking at it; a very long-term chronic infection kept at a completely subclinical level. This might be the wayan individual maintains immunity for long periods-even for a lifetime. " Lazzarini says that not much is known about immunologic memory-why once individuals are vaccinated they remain immune. Is it that the immune system, once stimulated, continuously produces antibodies, or does it require continued stimulation by a chronic subclinical infection? What is clear is that in infections induced by virus vaccine strains, one often finds evidence of the presence of these OI particles, and this may be the way a vaccine works; debilitating the virus, as it were, and thus limiting viral replication. This relatively new area of research has opened a whole new realm of possibilities in the fight against viral diseases.
-Charles S. Marwick American Pharmacy correspondent
15
DI Particles: Painting a Clearer Picture of Viruses and Vaccines
T
he neat picture of the classic viral infection-virus entering the host cell, the taking over of the cell's machinery to manufacture uniform progeny virus which, in turn, enter other cells to continue the process until halted by the host's developing antibody to the virus-is undergoing some major rethinking. Hand-in-hand with this changing concept are emerging some novel ideas regarding the way virus replication occurs at the molecular level, the nature and control of viral infections, and the way in which vaccines work to protect against viral diseases. I This is the outcome of current research into viral infections and, in particular, into the formation of what are known as defective interfering particles-fragments of the virus that arise spontaneously as viruses infect and reproduce within the host cell. Defective interfering particles, or 01 particles as they are called, have been found in almost every virus in which they have been sought, according to Dr. Robert A. Lazzarini, acting chief of the laboratory of molecular genetics at the National Institute of Neurological and Communicative Disorders and Stroke at the National Institutes of Health. Although these particles contain all the viral proteins, they contain only a small piece of the viral chromosome-often less than one gene-so they can't carry out all the functions of the complete virus. They are much smaller than the complete parent virus. But in all their morphological features, they are exactly like the whole virus. And, despite their defects, when a cell in which they are present is infected with the parent virus, they can be replicated," Lazzarini observes. When DI particles are replicated, it is done at the expense of the fully II
14
infective parent virus. Thus their presence in a cell reduces the number of whole infective virus particles assembled in the cell. In the case of vesicular stomatitis virus (VSV), for example, a cell that is infected with a single virus would produce about 10,000 progeny particles. But if DI particles are in that cell as well, it may yield only 10 or 100 particles of the complete virus. Thus, DI particles are not only defective-they also exert an interfering effect on virus infection. "In fact," Lazzarini points out, "if you have enough 01 particles in an infected cell, you can snuff out the virus infection completely in that cell. We have also found that the 01 particles produced in these cells will penetrate and similarly protect other cells in the face of a similar infection ." Lazzarini and his staff do most of their work with VSV because it is handled easily and obtainable in large quantities. It is similar in shape to rabies virus, looking a little like a bacillus, sort of bullet-shaped. 01 particles are extremely specific, Lazzarini told American Pharmacy. A 01 particle from VSV will only inhibit replication of VSV; it won't inhibit any other type of virus.
Relatively Benign. Most 01 particles don't appear to have any effect on the host cell. "If you take a cell and add 1,000 or 10,000 DI particles nothing happens to the host cell that we can see," Lazzarini said. "The cell still goes on multiplying in the normal way. There are some exceptions; some 01 particles will kill the cell, but they are rare." In about 1970, Drs. Alice Huang and David Baltimore speculated that the inhibiting effect of 01 particles on cell replication played a role in the control of natural infections-in fact terminating them. The particles,
they held, acted as natural, virusspecific antiviral agents. They also speculated that 01 particles might be responsible for chronic virus infections by interfering with the replication of the full, infectious virus. This prevented what would otherwise have been an acute infection, they reasoned . "We know something about the origin of 01 particles," Lazzarini said. "In essence they arise because of a mistake in replication. As the parent virus is replicated in the cell and its genome is being copied, the enzyme (replicase) that copies the genome makes a mistake. But it is a curious kind of mistake. While a substantial amount of information is left out, the essential information for copying remains. You can't just take a fragment of a virus chromosome and expect it to be copied; the enzyme doesn't know where to start because it doesn't have the proper ends. But a DI particle does, and so it is copied. "If one is looking to make use of such information in therapy or prevention of viral diseases, a reasonable approach would be to find I chemicals that might stimulate such mistakes in replication," Lazzarini continued. "If these can be found, then we have a way of creating the antiviral component in situ; right in the very cells that are infected with the virus. These chemicals would be very specific and not likely to inter- I fere with any host cell functions because the host has a completely different kind of replication machinery than do most viruses. There is a certain aesthetic appeal in targeting antiviral therapy in the specific cell i tha t is infected." Lazzarini has not pursued this approach. . "We have rather limited resources here," he says, and we're mostly interested in the basic scientific as' pects." II
American Pharmacy Vol. NS21 , No. 10, October 1981/5
Possible Vaccines However, he does speculate free-
ly about ways in which OI particles could be used to control viral infections: purified populations of OI particles as a type of vaccine for instance. "The particles aren't infectious, they carry a full variety of all the virus proteins with them, they penetrate cells, and would stimulate the immune system. There's no reason to believe they aren't immunogenic," he says. Right now Lazzarini and his asso-
useful in making a vaccine." Lazzarini outlines one possibility: "Suppose it were possible to make a hybrid virus out of a fully attenuated vaccine strain by inserting a gene of a second virus into its chromosome. If the inserted piece were positioned properly so that it were expressed as protein, the hybrid vaccine virus might stimulate an immune response to both parent viruses. "Great care would have to be taken to ensure that the hybrid virus did not acquire any detrimental biological properties. Using this strat-
Drs. Lazzarini and Manfried Schubert discuss an autoradiogram of eiectrophoretically separated radioactive 01 particle nucleic acids.
ciates are looking into gene splicing techniques as an avenue that may be useful not only in producing a vaccine, but may throw light on the gene sequences that are of importance in OI particles-possibly of viruses as well. With the aid of recombinant DNA procedures they are building OI particles by copying the appropriate sequences. "Once they're in DNA, they can be easily manipulated and we can glue them together, rearrange them and so forth," he says. "We're interested in this approach because it enables us to demonstrate what sequences are important and what are not. On a possibly more practical level, we might be able to splice into a virus chromosome information that would, if it were expressed, be ~~e" [nCan Pharmacy Vol.
NS21 , No. 10, October 1981 /567
egy, it may be possible not only to produce multipurpose vaccines but to produce vaccines against viruses not easily 'tamed' or attenuated." It is also from Lazzarini's laboratory that some provocative findings are emanating on the way vaccines work. Studies with viruses that have been attenuated in the laboratory by adapting them to growth in chick embryo cells-a standard method of manufacturing vaccines-is throwing light on the role of OI particles in the way a vaccine confers immunity. One of Lazarrini's associates, Dr. Micheline McCarthy, working with a chick embryo adapted strain of mumps virus, has shown that, while this virus can infect mammalian cells, the infection is inapparent. The cells appear microscop-
ically normal. But on close study, one sees that there is mumps virus being replicated in these cells and that this infection is completely dominated by OI particles. "Now," says Lazzarini, "if you take this attenuated strain of virus and put it into chick embryo cells, then there is no evidence of OI particles, but one gets a fulminating, full-blown viral infection. Again, if one takes other strains of mumps virus that have not been adapted to chick embryo cells and puts these into mammalian cell cultures then one again sees a fulminating infection and no evidence of the presence of 01 particles. "The point is that where there are 01 particles present, there seems to be attenuation. It may be that attenuation by OI particles is common among vaccine strains," Lazzarin i maintains . "It may be that what on e wants in a vaccine is a virus that only gives a subclinical infection so that it doesn't cause disease, but also permits viral replication to go forward so that the immune response is kept in a constant state of stimulation. "That's one way of looking at it; a very long-term chronic infection kept at a completely subclinical level. This might be the wayan individual maintains immunity for long periods-even for a lifetime. " Lazzarini says that not much is known about immunologic memory-why once individuals are vaccinated they remain immune. Is it that the immune system, once stimulated, continuously produces antibodies, or does it require continued stimulation by a chronic subclinical infection? What is clear is that in infections induced by virus vaccine strains, one often finds evidence of the presence of these OI particles, and this may be the way a vaccine works; debilitating the virus, as it were, and thus limiting viral replication. This relatively new area of research has opened a whole new realm of possibilities in the fight against viral diseases.
-Charles S. Marwick American Pharmacy correspondent
15