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Molecular Genetics of the Rotaviruses
Conference Report the era before Pasteur is followed by a description of the disease and the disease causing organism. The different vaccines are then described followed by a section on side effects and any contraindications. The social effects of using the vaccine and the epidemiology of the disease resulting from the widespread use of the vaccine is also common to most of the chapters. Wherever appropriate there is a full discussion of the relative merits of the different kinds of vaccine that are available and there are also some indications as to the kinds of new vaccines which are waiting in the wings, some in clinical trial while others have not reached that stage of maturity. If there are shortcomings it is in the science and technology of vaccines. Indeed the book is oriented to medical personnel who are at the interface between the manufacturer of vaccines and the recipient of those vaccines. The immunological problems and conundra, which the somewhat empirically derived vaccines have overcome remain to be sorted out. Additionally there is little presented about the new adjuvants, which promise much in terms of immunoprophylaxis but
which still need further work to realize their full potential. Again the ways in which the vaccines are produced was not dealt with in detail although it may be possible to pick up some of the pieces from the odd reference to the production techniques which was provided even though some of these references were to works, which themselves did not purport to tell the whole story. Viruses and bacteria loom large in these pages. Protozoa, helminths and fungi are hardly mentioned. This is not surprising as vaccines protective against diseases caused by these organisms have not been licensed in the USA. Nevertheless in a short chapter on recent developments that are likely to give rise to vaccines in the future there is a focus on the use of genetic engineering techniques, which can be justified as one of the most recently licensed vaccines, that protective against hepatitis B virus, can be derived from a genetically engineered yeast celt. Although the human immunodeficiency virus rated a chapter to itself, in spite of the distance between present knowledge and a licensed product it is salutary to reflect that most success in attempts to raise a vaccine
protective against the AIDS syndrome seems to have been achieved with a conventional killed virus vaccine in experiments, which have been done with the related Simian virus. From a somewhat erratic recounting of where we were some 100-200 years ago to the clear, full and valuable summary of what vaccines are available, including many details of their strengths and weaknesses, we have in this book a solid foundation from which we can carry the subject forward. It is an ideal text for those in the medical-public health fields and for those who from time to time have to educate students in the intricacies of vaccinology. There is yet room for further works on the immunological aspects of vaccines and the techniques by which they are produced and for a work of equivalent quality in the area of veterinary vaccines. In setting up these requirements and by providing an example resplendent in virtue the editors and contributors to this volume have given great service to the area of endeavour to which they and the readers are committed.
R.E. Spier
Conference Report Molecular Genetics of the Rotaviruses 20-21 April 1989, Jouy en Josas, France
Rotaviruses are the major agents responsible for infantile diarrhoea among humans as well as among animals. Since the beginning of the 1980s, knowledge of these viruses has accumulated considerably with respect to epidemiology and molecular biology. This first international meeting devoted solely to the molecular biology of the rotaviruses was organized in order to bring together recent results permitting new approaches to vaccination. This report presents the most striking points brought out in the meeting and emphasizes those problems that merit resolution in the near future.
Molecular structure and replication New methods of electron microscopy have permitted refinement of structural information on the virus particle and in particular have enabled determination of the position of gp4 on the capsid (V. Prasad). However, the nature of the
294 Vaccine, Vol. 8, June 1990
internal structure (core) is still very imprecise. Of the 11 segments of the genome, 10 have been sequenced. The analysis of the sequences and their comparison with sequence data banks permits predictions of functional sites, which show that the rotaviruses have diverged for a long time from the reoviruses with which they present very little sequence homology (J. Cohen). Correspondence between gene and product has now been established, particularly as the protein coded by gene number 3 has recently been identified. The nonstructural character of certain of the viral proteins has been defined, e.g. the product of gene 11 is non-structural, phosphorylated and undergoes post-translational modifications not yet elucidated (Estes). Several new findings define the steps of viral replication. An electrophoretic method which permits separation of nuclear protein complexes has been used to identify a series of sub-viral particles.
These particles are present on infected cells during the very early steps of morphogenesis, when the activity of the replicase is manifested (J. Patton). The mechanism of morphogenesis has been reconstituted in vitro, permitting the identification of the proteins that constitute the replicase. The problems related to the processing of viral proteins have been approached for the product of gene 11 and in particular for the vp7 glycoprotein. The peptide signal of vp7 (H2) plays a role both in targeting and in the maintenance of vp7 in the endoplasmic reticulum but the downstream sequence itself plays a role in maintaining vp7 in the RER. By modifying the anchor sequences it is possible to increase the immunogenicity of the recombinant protein (vp7 of rotavirus in a Vaccinia vector) (G. Both). In order to provide proteins that can be used as vaccine antigens, and to analyse the functions of viral genes, several viral proteins have been expressed in the form of recombinant proteins. The baculovirus system has been used for the greatest number of viral proteins, but Escherichia coil (0. and T. Nakagomi) and Vaccinia vectors (G. Both, M. Bremont), which
Conference Report allow the analysis of immune response to a replicating antigen, have also been used. In the case of E. coli, the expression of vp7 under the control of promoter omp-b, allows the expression of antigen at the surface of bacteria, which increases their immunogenicity. The epitopic analysis of the vp4 of SA11 shows a strong antigen at amino acids 95 to 103 (H. Streckert). Several groups have shown that vp-4 expressed in baculovirus binds specifically to MA104 cells and thus, without doubt, attaches to cellular receptors (E. Mackow, R. Bellinzoni); however, it seems that vp7 also can be recognized by cells (L. Babiuk). These results should soon make it possible to characterize the cellular receptors for virus infection. The product of gene 10, which is a non-structural protein, has also been expressed in baculovirus. It apparently acts as a receptor for the single capsid rotavirus particles and, therefore, plays a role in the final steps of morphogenesis (R. Bellinzoni).
Molecular epidemiology, virulence and genetics A number of questions concerning the epidemiology of rotaviruses are unresolved, including the reasons for seasonality and for the emergence of new strains (F. Clark). The selective pressures for intratypic and intertypic variation are unknown. J. Flores presented evidence that variation occurs both by genetic reassortment between strains and by intragenic mutations. G. Beards has serotyped nearly 1000 rotaviruses from all over the world and finds that 55% are serotype 1, 15% serotype 2, 13% serotype 3, 12% serotype 4 and the remaining 5% none of these types. A new mechanism of genetic evolution by rearrangement of genomic segments has recently been added to the available mechanisms of reassortment and point mutations. Until now, the rearrangements that have been demonstrated only related to a single genomic segment. A model explaining these rearrangements by jumps of the transcriptase on a segment in the course of transcription has been presented. This mechanism of rearrangement seems particularly important for the non-structural proteins. In the course of such rearrangements the product of the
gene can either be modified (gene 5) or not modified (gene 1I) (U. Desselberger and McCrae). Analysis of reassortants constitutes, for the rotaviruses and for other viruses with segmented genomes, an extremely powerful tool for the study of different genes. However, the results bearing on gene 4 remind us that a single gene expresses itself on the background of other genes. Some virus reassortants containing the same gene 4 in two different backgrounds are not recognized by monoclonal antibodies directed against vp4 (F. Ramig). Moreover, one observes few reassortants between the viruses of subgroups 1 and 2, as if the mixture of genes from the same subgroup has an internal coherence and provides a selective advantage (R. Ward). Gene 4 has already been shown in vitro and in the mouse model to play a role in the manifestation of virulence. However, the analysis of reassortants obtained from viruses that are virulent or avirulent for ten-day-old calves does not allow a strict correlation between a particular gene and virulence. While the virulent strains multiply to the same level as the avirulent strains, certain reassortants do not infect the calf but nevertheless induce seroconversion and protection (J. Bridger, D. Pocock). Not all animal rotaviruses have been serotyped. In cows, only two thirds of rotaviruses belong to the well characterized serotype 6 (D. Snodgrass). Horses appear to be predominantly serotype 3 viruses (G. Browning).
Mechanisms of protection, immune response, antigenic characterization, serotypes It is now generally agreed that the two proteins vp4 and vp7 carry epitopes that induce neutralizing antibodies, but doubt remains as to the capacity of vp6 to induce neutralizing antibodies. The consensus of the meeting was to designate the serotype of a strain by the use of a two-letter code, G and P, in order to note the specificities carried respectively by vp7 and vp4 (H. Greenberg). The antigenic characterization of certain strains illustrates the difficulty that exists in defining the relationship of an isolate to a single serotype when there is limitation on the antigens borne by glycoprotein vp7. A particular isolate
that was analysed for the sequences that code for the determinants of vp7 showed that this region was a structural mosaic containing residues coming from two serotypes. Such examples may indicate that the serotypes are not constituted discretely but are a continuum (I. Holmes). The use of monoclonal antibodies has made possible the definition of major group antigenic determinants and subgroup determinants carried by vp2 and vp6 (L. Svensson, P. Pothier). The preparation of polyclonal antibodies from recombinant proteins can also lead to useful reagents for the antigenic characterization of strains (H. Greenberg). In order to simplify the difficulties of classification, the concept of genogroup has been created. It is based on the genomic analogy between isolates and reference strains. This analogy is evaluated either globally by hybridization or locally by comparison of the sequence of a given gene (O. and T. Nakagomi, M. Gorziglia). After having been treated as controversial, the existence of heterotypic protection is now largely recognized and is one of the bases of vaccination attempts in infants. The analysis of the immune response to vaccines also demonstrates a dissociation between antibodies and protection. That dissociation suggests that defense mechanisms other than neutralizing antibodies play a role in the course of natural infection (F. Clark). The evidence of a CTL type response in a mouse model could also be an explanation for the protection observed in the absence of antibodies (P. Offit). The analysis of the immune response has been made easier by the use of ELISA for serotyping and subgrouping, combined with characterization of the immune response, and it is possible to show that both primary and secondary responses lead sometimes to a heterotypic conversion (G. Gerna, H. Brossow). The volume of work now being done on the structure and immune responses to rotaviruses induces optimism that vaccines can be developed, either by finding living viruses to stimulate broad mucosal immunity or by sensitizing the gut to particular prolative epitopes.
Jean Cohen Bernard Meignier Stanley A. Plotkin
Vaccine, Vol. 8, June 1990 295
which still need further work to realize their full potential. Again the ways in which the vaccines are produced was not dealt with in detail although it may be possible to pick up some of the pieces from the odd reference to the production techniques which was provided even though some of these references were to works, which themselves did not purport to tell the whole story. Viruses and bacteria loom large in these pages. Protozoa, helminths and fungi are hardly mentioned. This is not surprising as vaccines protective against diseases caused by these organisms have not been licensed in the USA. Nevertheless in a short chapter on recent developments that are likely to give rise to vaccines in the future there is a focus on the use of genetic engineering techniques, which can be justified as one of the most recently licensed vaccines, that protective against hepatitis B virus, can be derived from a genetically engineered yeast celt. Although the human immunodeficiency virus rated a chapter to itself, in spite of the distance between present knowledge and a licensed product it is salutary to reflect that most success in attempts to raise a vaccine
protective against the AIDS syndrome seems to have been achieved with a conventional killed virus vaccine in experiments, which have been done with the related Simian virus. From a somewhat erratic recounting of where we were some 100-200 years ago to the clear, full and valuable summary of what vaccines are available, including many details of their strengths and weaknesses, we have in this book a solid foundation from which we can carry the subject forward. It is an ideal text for those in the medical-public health fields and for those who from time to time have to educate students in the intricacies of vaccinology. There is yet room for further works on the immunological aspects of vaccines and the techniques by which they are produced and for a work of equivalent quality in the area of veterinary vaccines. In setting up these requirements and by providing an example resplendent in virtue the editors and contributors to this volume have given great service to the area of endeavour to which they and the readers are committed.
R.E. Spier
Conference Report Molecular Genetics of the Rotaviruses 20-21 April 1989, Jouy en Josas, France
Rotaviruses are the major agents responsible for infantile diarrhoea among humans as well as among animals. Since the beginning of the 1980s, knowledge of these viruses has accumulated considerably with respect to epidemiology and molecular biology. This first international meeting devoted solely to the molecular biology of the rotaviruses was organized in order to bring together recent results permitting new approaches to vaccination. This report presents the most striking points brought out in the meeting and emphasizes those problems that merit resolution in the near future.
Molecular structure and replication New methods of electron microscopy have permitted refinement of structural information on the virus particle and in particular have enabled determination of the position of gp4 on the capsid (V. Prasad). However, the nature of the
294 Vaccine, Vol. 8, June 1990
internal structure (core) is still very imprecise. Of the 11 segments of the genome, 10 have been sequenced. The analysis of the sequences and their comparison with sequence data banks permits predictions of functional sites, which show that the rotaviruses have diverged for a long time from the reoviruses with which they present very little sequence homology (J. Cohen). Correspondence between gene and product has now been established, particularly as the protein coded by gene number 3 has recently been identified. The nonstructural character of certain of the viral proteins has been defined, e.g. the product of gene 11 is non-structural, phosphorylated and undergoes post-translational modifications not yet elucidated (Estes). Several new findings define the steps of viral replication. An electrophoretic method which permits separation of nuclear protein complexes has been used to identify a series of sub-viral particles.
These particles are present on infected cells during the very early steps of morphogenesis, when the activity of the replicase is manifested (J. Patton). The mechanism of morphogenesis has been reconstituted in vitro, permitting the identification of the proteins that constitute the replicase. The problems related to the processing of viral proteins have been approached for the product of gene 11 and in particular for the vp7 glycoprotein. The peptide signal of vp7 (H2) plays a role both in targeting and in the maintenance of vp7 in the endoplasmic reticulum but the downstream sequence itself plays a role in maintaining vp7 in the RER. By modifying the anchor sequences it is possible to increase the immunogenicity of the recombinant protein (vp7 of rotavirus in a Vaccinia vector) (G. Both). In order to provide proteins that can be used as vaccine antigens, and to analyse the functions of viral genes, several viral proteins have been expressed in the form of recombinant proteins. The baculovirus system has been used for the greatest number of viral proteins, but Escherichia coil (0. and T. Nakagomi) and Vaccinia vectors (G. Both, M. Bremont), which
Conference Report allow the analysis of immune response to a replicating antigen, have also been used. In the case of E. coli, the expression of vp7 under the control of promoter omp-b, allows the expression of antigen at the surface of bacteria, which increases their immunogenicity. The epitopic analysis of the vp4 of SA11 shows a strong antigen at amino acids 95 to 103 (H. Streckert). Several groups have shown that vp-4 expressed in baculovirus binds specifically to MA104 cells and thus, without doubt, attaches to cellular receptors (E. Mackow, R. Bellinzoni); however, it seems that vp7 also can be recognized by cells (L. Babiuk). These results should soon make it possible to characterize the cellular receptors for virus infection. The product of gene 10, which is a non-structural protein, has also been expressed in baculovirus. It apparently acts as a receptor for the single capsid rotavirus particles and, therefore, plays a role in the final steps of morphogenesis (R. Bellinzoni).
Molecular epidemiology, virulence and genetics A number of questions concerning the epidemiology of rotaviruses are unresolved, including the reasons for seasonality and for the emergence of new strains (F. Clark). The selective pressures for intratypic and intertypic variation are unknown. J. Flores presented evidence that variation occurs both by genetic reassortment between strains and by intragenic mutations. G. Beards has serotyped nearly 1000 rotaviruses from all over the world and finds that 55% are serotype 1, 15% serotype 2, 13% serotype 3, 12% serotype 4 and the remaining 5% none of these types. A new mechanism of genetic evolution by rearrangement of genomic segments has recently been added to the available mechanisms of reassortment and point mutations. Until now, the rearrangements that have been demonstrated only related to a single genomic segment. A model explaining these rearrangements by jumps of the transcriptase on a segment in the course of transcription has been presented. This mechanism of rearrangement seems particularly important for the non-structural proteins. In the course of such rearrangements the product of the
gene can either be modified (gene 5) or not modified (gene 1I) (U. Desselberger and McCrae). Analysis of reassortants constitutes, for the rotaviruses and for other viruses with segmented genomes, an extremely powerful tool for the study of different genes. However, the results bearing on gene 4 remind us that a single gene expresses itself on the background of other genes. Some virus reassortants containing the same gene 4 in two different backgrounds are not recognized by monoclonal antibodies directed against vp4 (F. Ramig). Moreover, one observes few reassortants between the viruses of subgroups 1 and 2, as if the mixture of genes from the same subgroup has an internal coherence and provides a selective advantage (R. Ward). Gene 4 has already been shown in vitro and in the mouse model to play a role in the manifestation of virulence. However, the analysis of reassortants obtained from viruses that are virulent or avirulent for ten-day-old calves does not allow a strict correlation between a particular gene and virulence. While the virulent strains multiply to the same level as the avirulent strains, certain reassortants do not infect the calf but nevertheless induce seroconversion and protection (J. Bridger, D. Pocock). Not all animal rotaviruses have been serotyped. In cows, only two thirds of rotaviruses belong to the well characterized serotype 6 (D. Snodgrass). Horses appear to be predominantly serotype 3 viruses (G. Browning).
Mechanisms of protection, immune response, antigenic characterization, serotypes It is now generally agreed that the two proteins vp4 and vp7 carry epitopes that induce neutralizing antibodies, but doubt remains as to the capacity of vp6 to induce neutralizing antibodies. The consensus of the meeting was to designate the serotype of a strain by the use of a two-letter code, G and P, in order to note the specificities carried respectively by vp7 and vp4 (H. Greenberg). The antigenic characterization of certain strains illustrates the difficulty that exists in defining the relationship of an isolate to a single serotype when there is limitation on the antigens borne by glycoprotein vp7. A particular isolate
that was analysed for the sequences that code for the determinants of vp7 showed that this region was a structural mosaic containing residues coming from two serotypes. Such examples may indicate that the serotypes are not constituted discretely but are a continuum (I. Holmes). The use of monoclonal antibodies has made possible the definition of major group antigenic determinants and subgroup determinants carried by vp2 and vp6 (L. Svensson, P. Pothier). The preparation of polyclonal antibodies from recombinant proteins can also lead to useful reagents for the antigenic characterization of strains (H. Greenberg). In order to simplify the difficulties of classification, the concept of genogroup has been created. It is based on the genomic analogy between isolates and reference strains. This analogy is evaluated either globally by hybridization or locally by comparison of the sequence of a given gene (O. and T. Nakagomi, M. Gorziglia). After having been treated as controversial, the existence of heterotypic protection is now largely recognized and is one of the bases of vaccination attempts in infants. The analysis of the immune response to vaccines also demonstrates a dissociation between antibodies and protection. That dissociation suggests that defense mechanisms other than neutralizing antibodies play a role in the course of natural infection (F. Clark). The evidence of a CTL type response in a mouse model could also be an explanation for the protection observed in the absence of antibodies (P. Offit). The analysis of the immune response has been made easier by the use of ELISA for serotyping and subgrouping, combined with characterization of the immune response, and it is possible to show that both primary and secondary responses lead sometimes to a heterotypic conversion (G. Gerna, H. Brossow). The volume of work now being done on the structure and immune responses to rotaviruses induces optimism that vaccines can be developed, either by finding living viruses to stimulate broad mucosal immunity or by sensitizing the gut to particular prolative epitopes.
Jean Cohen Bernard Meignier Stanley A. Plotkin
Vaccine, Vol. 8, June 1990 295