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Vaccine antigens carried by vaccinia viruses
IDN Volume 6, Number 6, June 1987
Editor
Paul D. Hoeprich, MD Division of Infectious and Immunologic Diseases University of California, Davis Medical Center
Associate Editors
Ruth M. Lawrence, MD
Larry K. Pickering, MD
Charles W. Stratton, MD
Division of Infectious Diseases Texas Tech University Health Sciences Center
Program in Infectious Diseases and Clinical Microbiology The University of Texas Medical School at Houston
Department of Pathology Vanderbilt University Medical Center
Vaccine Antigens Carried by Vaccinia Viruses Vaccine Antigens Carried by Vaccfnin VirUWS
Kathryn M. Edwards, MD Division of Infectious Diseases, Department of Pediatrics, Vanderbilt University, Nashville, Tennessee 43
4s
47
The prevention of infectious diseases by immunization is a major goal of medical science. A prime example of attaining this goal was the global eradication of smallpox, a feat that was also testimony to the utility of the immunizing agent, vaccinia virus. In addition to proved immunogenic efficacy, vaccinia virus has historical and personal familiarity-most physicians have been inoculated and have inoculated others ivith it. Hence, serious consideration of the use of vaccinia virus as a carrier or vector for vaccine antigens reasonably followed demonstration of protection of laboratory animals immunized with vaccinia recombinants expressing foreign genes encoding bacterial, viral, or protozoa1 antigens.
Characteristicsof Vaccinia Virus
Elsevier 0278-2316/87/SO.O0
+ 2.20
Vaccinia virus is a member of the orthopoxvirus genus. Other members of this genus include smallpox, cowpox, ectromelia (mousepox), rabbitpox, and mon-
keypox. The origins of vaccinia virus are not certain, but it may have been derived from smallpox, cowpox, or horsepox virus. Vaccinia virus is not found in nature but has been maintained for many years in laboratories throughout the world. Vaccinia virus is a large, doublestranded DNA virus containing 187 kbp.* The length of the genome can, however, vary greatly while still retaining its ability to replicate. Viable 9 kbt deletion mutants of vaccinia virus have been isolated and mutants containing 25 kb insertions into the virus retain infectivity. The ability of different lengths of DNA to be packaged may relate to the nonicosahedral, complex structure of vaccinia virus. Because of its unique ability to tolerate variability in genomic size, vaccinia virus is an attractive vector. In addition, some vaccinia virus genes are nonessential for replication and *Kbp = Kilobase pairs; tKb = Kilobase.
0278-2316 IDINDN6(6)
43-50, 1987
44 Infectious Diseases Newsletter 6(6) June 1987
may be interrupted by genes from other infectious agents. The thymidine kinase (TIC) gene comprises one such nonessential region. Insertion of foreign DNA within the sequence of the vaccinia TK gene generates TK mutants that may be selected easily by preferential growth characteristics; they retain full infectivity. Finally, the vaccinia virus recombinants, like native virus itself, replicate in the cytoplasm of the infected cell and remain intracellular. Gene products of the recombinant-infected cells are, however, glycosylated and transported to the cell surface where they may be recognized by the host’s immune system. Construction of Vaccinia Recombiants The nucleotide sequences of vaccinia virus promoters are unique and are required immediately upstream of DNA to be transcribed. Thus, the initial step in generating vaccinia recombinants involves combining the vaccinia promoter to the coding sequences of the foreign gene that is to be expressed. The attachment of the vaccinia promoter with foreign gene sequences is accomplished by the use of a plasmid vector also termed an insertion vector (Figure 1). The insertion vector contains: 1) a vaccinia virus promoter; 2) unique restriction endonuclease cleavage sites downstream from the promoter; 3) vaccinia DNA sequences from a gene that is nonessential for viral replication (TK gene) that flanks the promoter and restriction sites, enabling the plasmid to insert itself by homologous recombination into the viral genome; and 4) selection markers to allow selection of recombinant virus-infected cells. To insert the foreign gene sequence, the insertion vector is first cleaved by an appropriate restriction endonu-
Figure 1. Construction of vaccinia virus vectors expressing foreign genes. Adapted from Smith and Moss.
clease, then the foreign gene is inserted and ligated into the vector. The resulting recombinant is then introduced into cells infected with wild-type vaccinia virus. Recombination takes place with insertion of the foreign gene into the vaccinia genome. The recombinants are then selected by their TK-deficient growth characteristics. The recombinant viruses have foreign DNA integrated into the vaccinia genome without deletions or rearrangements. They retain their infectivity since no essential functions are deleted. Insertion of as many as 25 kbp has been accomplished into the vaccinia virus. The use of deletion mutants of vaccinia virus could increase the capacity for additional DNA up to 35 kbp. In summary, genes from virtually any infectious agent may be incorporated into the vaccinia genome. Prospects for the Use of Vaccinia Virus Vectors The global eradication of smallpox demonstrated the effectiveness of vaccinia as an immunizing agent.
Several attributes of the vaccinia virus made it an ideal antigen: 1. Ease of administration. Vaccinia virus is easily administered to large numbers of people with little equipment and briefly trained personnel. The use of a simple, bifurcated needle, introduced in 1968, enabled workers with limited medical training to flame-sterilize or boil needles; a single needle sufficed for several hundred inoculations. 2. Vaccine production. Local production of vaccine reduced the costs. Both the World Health Organization and the United Nations Children’s Educational Fund supported laboratories in developing countries for vaccine production. Calves or water buffalo were scarified with vaccinia virus and large quantities of virus were harvested, purified, and freezedried. Thus, the costs of vaccine production were minimal. 3. Vaccine stability. Freeze-dried vaccinia virus retains its infectivity without refrigeration, enabling the vaccine to be used in remote, primitive areas. 4. Vaccine efficacy. Remarkable vaccine efficacy in preventing smallpox in endemic areas was clearly demonstrated in a report in 1971. Ninety percent of the cases of smallpox occurred in individuals with no vaccination scar and no history of vaccination. Many persons who were partially protected by immunization experienced subclinical cases of smallpox when challenged naturally. One further study in an endemic area documented vaccine efficacy of greater than 80% in those vaccinated at least 20 years previously. Similar data are not available from nonendemic areas. 5. Stimulation of both humoral and cell-mediated immunity; after primary intradermal inoculation of the (conrimed
on page 48)
lnfeoious Discorer Newsletter fISSN 02%2316) is issued monrhlv in one Indexed volume uer year bv Elsevier Science Publishing Co.. Inc.. 52 Vanderbilt Avenue. New York. NY 10017 Printed in USA at 25 Sand Creek Road, Albany. NY 12205. Subs&don priceper yea;: instmnions. 5112.00:m&id& i56.00. For an mail to Europeladd $21.00: for an mall elsewhere. add $24.00. Second-classpostagepmding ar New York. NY. and at additional mailing offices Postmasrer~Send addresschangesto Inferrims Dirroxs Newxkrfer, Elscvicr Science Publtshmg Co.. Vanderbrlt Avenue. New York. NY 10017.
Inc 52
@
1987 Elsevier Science Publishing 0278-2316/87/%0.00
+ 2.20
Co., Inc.
Editor
Paul D. Hoeprich, MD Division of Infectious and Immunologic Diseases University of California, Davis Medical Center
Associate Editors
Ruth M. Lawrence, MD
Larry K. Pickering, MD
Charles W. Stratton, MD
Division of Infectious Diseases Texas Tech University Health Sciences Center
Program in Infectious Diseases and Clinical Microbiology The University of Texas Medical School at Houston
Department of Pathology Vanderbilt University Medical Center
Vaccine Antigens Carried by Vaccinia Viruses Vaccine Antigens Carried by Vaccfnin VirUWS
Kathryn M. Edwards, MD Division of Infectious Diseases, Department of Pediatrics, Vanderbilt University, Nashville, Tennessee 43
4s
47
The prevention of infectious diseases by immunization is a major goal of medical science. A prime example of attaining this goal was the global eradication of smallpox, a feat that was also testimony to the utility of the immunizing agent, vaccinia virus. In addition to proved immunogenic efficacy, vaccinia virus has historical and personal familiarity-most physicians have been inoculated and have inoculated others ivith it. Hence, serious consideration of the use of vaccinia virus as a carrier or vector for vaccine antigens reasonably followed demonstration of protection of laboratory animals immunized with vaccinia recombinants expressing foreign genes encoding bacterial, viral, or protozoa1 antigens.
Characteristicsof Vaccinia Virus
Elsevier 0278-2316/87/SO.O0
+ 2.20
Vaccinia virus is a member of the orthopoxvirus genus. Other members of this genus include smallpox, cowpox, ectromelia (mousepox), rabbitpox, and mon-
keypox. The origins of vaccinia virus are not certain, but it may have been derived from smallpox, cowpox, or horsepox virus. Vaccinia virus is not found in nature but has been maintained for many years in laboratories throughout the world. Vaccinia virus is a large, doublestranded DNA virus containing 187 kbp.* The length of the genome can, however, vary greatly while still retaining its ability to replicate. Viable 9 kbt deletion mutants of vaccinia virus have been isolated and mutants containing 25 kb insertions into the virus retain infectivity. The ability of different lengths of DNA to be packaged may relate to the nonicosahedral, complex structure of vaccinia virus. Because of its unique ability to tolerate variability in genomic size, vaccinia virus is an attractive vector. In addition, some vaccinia virus genes are nonessential for replication and *Kbp = Kilobase pairs; tKb = Kilobase.
0278-2316 IDINDN6(6)
43-50, 1987
44 Infectious Diseases Newsletter 6(6) June 1987
may be interrupted by genes from other infectious agents. The thymidine kinase (TIC) gene comprises one such nonessential region. Insertion of foreign DNA within the sequence of the vaccinia TK gene generates TK mutants that may be selected easily by preferential growth characteristics; they retain full infectivity. Finally, the vaccinia virus recombinants, like native virus itself, replicate in the cytoplasm of the infected cell and remain intracellular. Gene products of the recombinant-infected cells are, however, glycosylated and transported to the cell surface where they may be recognized by the host’s immune system. Construction of Vaccinia Recombiants The nucleotide sequences of vaccinia virus promoters are unique and are required immediately upstream of DNA to be transcribed. Thus, the initial step in generating vaccinia recombinants involves combining the vaccinia promoter to the coding sequences of the foreign gene that is to be expressed. The attachment of the vaccinia promoter with foreign gene sequences is accomplished by the use of a plasmid vector also termed an insertion vector (Figure 1). The insertion vector contains: 1) a vaccinia virus promoter; 2) unique restriction endonuclease cleavage sites downstream from the promoter; 3) vaccinia DNA sequences from a gene that is nonessential for viral replication (TK gene) that flanks the promoter and restriction sites, enabling the plasmid to insert itself by homologous recombination into the viral genome; and 4) selection markers to allow selection of recombinant virus-infected cells. To insert the foreign gene sequence, the insertion vector is first cleaved by an appropriate restriction endonu-
Figure 1. Construction of vaccinia virus vectors expressing foreign genes. Adapted from Smith and Moss.
clease, then the foreign gene is inserted and ligated into the vector. The resulting recombinant is then introduced into cells infected with wild-type vaccinia virus. Recombination takes place with insertion of the foreign gene into the vaccinia genome. The recombinants are then selected by their TK-deficient growth characteristics. The recombinant viruses have foreign DNA integrated into the vaccinia genome without deletions or rearrangements. They retain their infectivity since no essential functions are deleted. Insertion of as many as 25 kbp has been accomplished into the vaccinia virus. The use of deletion mutants of vaccinia virus could increase the capacity for additional DNA up to 35 kbp. In summary, genes from virtually any infectious agent may be incorporated into the vaccinia genome. Prospects for the Use of Vaccinia Virus Vectors The global eradication of smallpox demonstrated the effectiveness of vaccinia as an immunizing agent.
Several attributes of the vaccinia virus made it an ideal antigen: 1. Ease of administration. Vaccinia virus is easily administered to large numbers of people with little equipment and briefly trained personnel. The use of a simple, bifurcated needle, introduced in 1968, enabled workers with limited medical training to flame-sterilize or boil needles; a single needle sufficed for several hundred inoculations. 2. Vaccine production. Local production of vaccine reduced the costs. Both the World Health Organization and the United Nations Children’s Educational Fund supported laboratories in developing countries for vaccine production. Calves or water buffalo were scarified with vaccinia virus and large quantities of virus were harvested, purified, and freezedried. Thus, the costs of vaccine production were minimal. 3. Vaccine stability. Freeze-dried vaccinia virus retains its infectivity without refrigeration, enabling the vaccine to be used in remote, primitive areas. 4. Vaccine efficacy. Remarkable vaccine efficacy in preventing smallpox in endemic areas was clearly demonstrated in a report in 1971. Ninety percent of the cases of smallpox occurred in individuals with no vaccination scar and no history of vaccination. Many persons who were partially protected by immunization experienced subclinical cases of smallpox when challenged naturally. One further study in an endemic area documented vaccine efficacy of greater than 80% in those vaccinated at least 20 years previously. Similar data are not available from nonendemic areas. 5. Stimulation of both humoral and cell-mediated immunity; after primary intradermal inoculation of the (conrimed
on page 48)
lnfeoious Discorer Newsletter fISSN 02%2316) is issued monrhlv in one Indexed volume uer year bv Elsevier Science Publishing Co.. Inc.. 52 Vanderbilt Avenue. New York. NY 10017 Printed in USA at 25 Sand Creek Road, Albany. NY 12205. Subs&don priceper yea;: instmnions. 5112.00:m&id& i56.00. For an mail to Europeladd $21.00: for an mall elsewhere. add $24.00. Second-classpostagepmding ar New York. NY. and at additional mailing offices Postmasrer~Send addresschangesto Inferrims Dirroxs Newxkrfer, Elscvicr Science Publtshmg Co.. Vanderbrlt Avenue. New York. NY 10017.
Inc 52
@
1987 Elsevier Science Publishing 0278-2316/87/%0.00
+ 2.20
Co., Inc.