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Live attenuated vaccines for human use
Live attenuated vaccines for human use
Stanley J. Cryz Jr. Swiss Serum and Vaccine Institute, Berne, Switzerland Live attenuated vaccines have been successfully used for the prevention of a number of viral and bacterial diseases. Several vaccine strains have been utilized recently as expression vectors for cloned heterologous antigens. Through the use of recombinant DNA technology, candidate vaccine strains and vector systems have been developed and are undergoing clinical evaluation. Current Opinion in Biotechnology 1992, 3:298-302
Introduction Several live vaccines directed against bacterial and viral pathogens are n o w licensed for h u m a n use (Table 1). The potential of live vaccines is best illustrated by the eradication of smallpox. In several countries, nearly complete immunization against polio, measles, mumps, and rubella has resulted in a dramatic decrease in disease incidence. Eradication of such diseases, where man is the sole natural reservoir, is theoretically possible.
Table 1. Live attenuated vaccines currently licensed for human use.
Disease
Vaccine
Year of introduction
Smallpox Naturally attenuated cowpox virus Tuberculosis Bacille Calmette-Gu~rin (BCG) Yellow fever 17 D strain Polio Trivalent, tissue culture passaged Measles Tissue culture passaged Mumps Tissue culture passaged Rubella Tissue culture passaged Typhoid fever Ty21 a strain Adenovirus Tissue culture passaged Japanese encephalitis Tissue culture passaged Varicella OKA strain, tissue culture passaged
1798 1927 1936 1961 1963 1967 1969 1980 1983 1983 1983
In spite of these successes, infectious diseases still exact a considerable toll worldwide. Vaccines are needed to control parasitic diseases and a number of enteric diseases caused by bacterial and viral pathogens. The application of recombinant DNA technology has led to the construction of numerous novel live vaccine candidates and vectors that hold great promise for the future [1-'].
A number of n e w attenuated vaccine candidates have b e e n developed, some of which are undergoing clinical evaluation (Table 2). This review will primarily focus u p o n the various techniques currently being explored in the development of live attenuated vaccine strains through the use of recombinant DNA technology.
Safety of live attenuated vaccines currently in use
Live vaccines have an outstanding benefit-to-risk ratio and serious post-immunization sequelae are rare. Bacille Calmette-Gu~rin (BCG) tuberculosis vaccine (the most widely used live vaccine with nearly 3 billion doses administered) has a case fatality ratio of roughly 2 per 10 million doses. Approximately 1 in 500 000 recipients of oral polio vaccine will develop poliomyelitis. Aseptic meningitis (non-paralytic polio) lasting 2 to 10 days is a more c o m m o n occurrence but the majority of individuals recover completely. Central nervous system (CNS) disorders following administration of measles vaccine occur at a rate of roughly 1 per 3 million doses. Serious adverse reactions following receipt of rubella or mumps vaccine are rare. These figures demonstrate that severe CNS complaints following immunizatidn with measles, mumps, or rubella vaccine appear at a rate comparable to the background level within the general population. A total of 15 cases of encephalitis has been temporally associated with the administration of 100 million doses of the 17D yellow fever vaccine. Approximately 15 million North American military recruits have received adenovirus vaccine without any substantial untoward effects. Nearly 100 million doses of Ty21a typhoid vaccine have been administered with no serious reaction noted. In summary, live attenuated vaccines rarely cause serious, irreversible, adverse reactions.
Abbreviations BCG--Bacille Calmette-Gu~rin; CMV---cytomegalovirus; CNS~central nervous system; HIV--human immunodeficiency virus; LPS--lipopolysaccharide; RSV--respiratory syncytial virus; TCID--tissue culture infective dose.
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Live attenuated vaccines for human use Cryz 299 I
[Table 2. Live attenuated vaccines under development. Disease
Vaccine
Route
Cholera
A lsubunit deletion mutant Vibrio cholerae CVD103-Hg Salmonella typhi Ty21a expressing V. cholerae O-anti
Oral Oral
Aromatic amino acid (ARO) mutants CRA, CRP deficient mutants Temperature-sensitive mutants
Oral Oral Oral
ARO mutants Escherichia coli-Shigella hybrid strains S. typhi Ty21a expressing 5higella sonnei O-antigen
Oral Oral Oral
E. coli diarrhea
ARO mutants Enterotoxi n-deficient mutants
Oral Oral
Influenza
Cold-adapted virus Avian-human reassortant virus Cold-adapted reassortant virus
Intranasal Intranasal Intranasal
Rotavirus
Rhesus rotavirus, attenuated by tissue culture passage Bovine rotavirus, attenuated by tissue culture passage Human rotavirus, attenuated by tissue culture passage
Oral Oral Oral
Hepatitis A
Attenuated by tissue culture passage
Parenteral
Cytomegalovirus
Attenuated by tissue culture passage
Parenteral
Dengue
Attenuated by tissue culture passage
Parenteral
Acquired immunodeficiency syndrome
Vaccinia virus expressing HIV antigens Sabin type 1 polio virus expressing HIV antigens BCG expressing HIV antigens
Parenteral Oral Parenteral or oral
Hepatitis B
Adenovirus expressing hepatitis B surface antigen
Parenteral
Typhoid fever
Shigellosis
Approaches to live vaccine development
tors, holds great promise for the construction of multivalent live vaccines [1"]. Alternatively, strains can be attenuated by the deletion or inactivation of genes with k n o w n virulence-enhancing attributes. Several such recombinant strains have been developed and are undergoing clinical evaluation.
With one exception, the Salmonella typhi Ty21a strain, all live vaccines currently in use were developed empirically. Attenuation of viral vaccines was accomplished by repeated passaging on tissue culture ceils, eggs, or a combination of the two. Several passage levels were then selected and tested in an effort to isolate an avirulent virus capable of sufficient replication in man to engender a protective immune response without attendant adverse reactions. The BCG vaccine strain was attenuated by serial passage of Mycobacterium bovis for 13 years. The two major drawbacks to such an approach are undefined attenuating mutation(s) and potential strain instability. Even so, these vaccines have been proven to be remarkably safe and effective. This approach continues to be used today in the development of new vaccine strains.
The use of live vaccine strains as vectors for candidate vaccine antigens is particularly attractive for several reasons: their safety has already been established; they have the potential to immunize against two or more disease entities; and recombinant strains hyper-producing one or more protective antigens might prove to be more effective as immunizing agents than the attenuated pathogen itself.
The application of recombinant DNA technology to live vaccine development has allowed for a more rational approach. The identification of critical protective antigens, followed by cloning and expression in live vec-
A number of foreign genes have been inserted into the smallpox vaccine (Vaccinia virus) [1"'-3"]. Several such recombinant strains have been shown to afford protection against bacterial and viral pathogens in ex-
Live vaccines based upon vector systems
300 Evironmentalbiotechnology perimental systems. O n e drawback to this approach in humans is the unacceptably high rate of reactions associated with the smallpox vaccine. To circumvent this problem, several genes have b e e n deleted to further attenuate this strain. A thymidine kinase-deficient variant expressing h u m a n immunodeficiency virus (HIV) gp 160 w a s found to be safe in HIV seronegative males [3"']. However, in subjects w h o had previously b e e n immunized against smallpox it engendered only a weak, transient anti-HIV immune response. In contrast, subjects w h o had not previously b e e n immunized m o u n t e d a vigorous, long lasting anti-HIV T-cell and antibody response. These results suggest that preexisting immunity to a vaccine vector m a y suppress the host's ability to respond to the recombinant antigen. The use of the BCG vaccine strain as a vector has m a n y potential advantages including its d o c u m e n t e d safety, low cost, and current widespread use as a childhood vaccine, as well as the ability of a single dose of the vaccine to induce long-lasting protection. Several recombinant BCG strains have b e e n constructed that stably express foreign genes, including several from IMV [4"',5]. Preliminary results in animals have demonstrated that these constructs can induce both humoral and cell-mediated i m m u n e responses. Genes encoding antigens from h u m a n papilloma virus, hepatitis A virus, influenza virus, Herpes simplex virus, HIV, respiratory syncytial virus (RSV), and coxsackie B virus have b e e n inserted into the g e n o m e of Sabin type 1 oral polio virus [1"']. Several of these recombinant strains have b e e n s h o w n to induce a neutralizing antibody response to both polio virus and the heterologous pathogen. However, in several instances, antibody was unable to recognize the native antigen as expressed b y the heterologous pathogen. Recombinant adenovirus type 4 and 7 strains expressing hepatitis B surface antigen have b e e n constructed [6]. Chimpanzees immunized by the oral route with these strains were protected against hepatitis B challenge. Several HIV genes and the gene encoding the F glycoprotein of RSV have also b e e n introduced into adenovirus [1"]. Immunization of cotton rats with the F glycoprotein recombinant protected against RSV challenge. As the S. typhi Ty21a strain has a remarkable safety record and proven efficacy [7"], it has b e e n utilized as a vector expressing the antigens of other enteric pathogens. The rfb genes of Shigella sonnei and Vibrio cholerae, which code for the O-antigen moiety of lipopolysaccharide (LPS), have b e e n introduced into Ty21a [8,9]. The S. typhi-S, sonnei hybrid provided a modest degree of protection in volunteers against a S. sonnei challenge [8]. However, efficacy varied from one batch to another.':Although the precise reason for this p h e n o m e n o n remains unknown, it m a y be related in part to the in vivo instability of the plasmid coding for the O-antigen, or to the fact that the O-antigen is not covalently linked to the core of S. typhi LPS. The S. typhi-V, cholerae hybrid contains the rfa gene encoding Escherichia coli LPS core and the rfb gene of V. cholerae, which allows for the attachment of the V.
cholerae O-antigen to the E. coli core. Multiple doses of this vaccine resulted in a 25 % reduction in the diarrhea attack rate following a V. cholerae challenge [9]. This modest protection was attributed to the low levels of antibody to V. cholerae O-antigen induced by the vaccine. Inactivation of the S. typhi u~b gene, which allows for m o r e efficient expression of the V. cholerae O-antigen on the cell surface, significantly increased the immune response in humans [10]. The efficacy of this strain is currently under evaluation.
Live candidate vaccines developed through recombinant DNA technology A live, oral, attenuated cholera vaccine termed CVD103-HgR has been developed b y deletion of the enzymatically active A 1 subunit of cholera toxin [11]. This strain has b e e n found to be safe and i m m u n o g e n i c in double-blind, randomized, placebo-controlled trials in North American adults, Swiss adults [12], Thai adults [13], and in Indonesian children between 2 and 9 years of age (MM Levine, personnal communication). A single orally administered dose of vaccine provided high levels of protection (62-100%) against experimental challenge with V. cholerae, which last for at least 6 months [11] (M Levine, unpublished data). E x p a n d e d Phase II safety and immunogenicity studies are n o w u n d e r w a y in Asia and South America as a prelude to field trials in cholera-endemic areas. Bacterial auxotrophic mutants with complete blocks in aromatic amino acid synthesis pathway (ARO) are avirulent in mammalian hosts because of their inability to produce or acquire p-aminobenzoic acid (a folic acid precursor) and 2,3-dihydroxybenzoic acid (a precursor of bacterial siderophores) from the host [14"]. A Shigellaflexneri aroD deletion mutant has b e e n found to be safe, immunogenic, and protective in m o n k e y studies [14"]. H o n e et al. [15] have constructed double aro mutants b y deletion of the aroC and D genes in S. typhi. One such strain, CVD908, has b e e n f o u n d to be safe and remarkably immunogenic in humans after a single oral dose (M Levine, personal c o m m u nication). ARO mutants m a y prove to be highly effective live oral vaccines against several enteric diseases because of their safety and apparent undiminished immunogenicity. These traits could also make t h e m ideal vectors for the delivery of heterologous antigens.
Live candidate vaccines developed by classical methods of attenuation T w o live strains of hepatitis A, attenuated b y passage on cultured cells, are currently undergoing clinical evaluation [16,17]. The H2 strain has b e e n parenterally administered to 12 healthy seronegative adults and 127 children, 4-12 years old at a tissue culture infective dose (TCID50) of 106.5 [16]. There was n o evidence of liver toxicity following vaccination and all subjects seroconverted. One potential drawback of this vaccine is that it is cultured on the KMB17 h u m a n lung fibroblast cell line, a substrate not routinely used for propaga-
Live attenuated vaccinesfor human use Cryz 301 tion of human vaccines. The F' variant of strain CR326F was also well tolerated w h e n parenterally administered to volunteers [17]. At a dose of 107.3 TCID50 , 10 out of 10 subjects seroconverted. Both vaccines induced longlived humoral immune responses and neutralizing antibodies to hepatitis A vires (assayed in vitro). Although such live attenuated vaccines were less immunogenic than inactivated hepatitis A vaccines administered by a multidose regimen, they may prove to be highly effective as a result of efficient priming of the immune system and the need for only a single dose. Several live attenuated rotavirus candidate vaccine strains have been developed and field tested [18"]. Results from Finnish and South American studies have shown highly significant protection. The same strains, however, were found to be ineffective when tested in other geographical areas [18"',19]. In some cases, lack of protection could be explained by the fact that the endemic field strains were of a heterologous serotype. In other trials, differences in efficacy could be related to the age of immunization. Protection was lower in infants, w h o generally mounted a poorer immune response. It n o w appears that a multi-serotype rotavims vaccine that is highly immunogenic in infants will be needed. The Towne live attenuated strain of cytomegalovirus (CMV) was derived after 105 passages o n h u m a n diploid ceils. Immunization of seronegative subjects resulted in partial protection following parenteral challenge with a low passage strain of CMV [20]. Naturally seropositive subjects were resistant to a 10-fold higher challenge dose of vires than the Towne strain recipients. Immunization of seronegative renal transplant recipients, w h o subsequently received an organ from a seropositive donor, resulted in a significant decrease in disease scores compared with a placebo group [21]. Graft survival was also higher in the vaccinated group. Immunization did not, however, influence the rate of infection. Several live-attenuated, cold-adapted reassortant influenza vaccine strains have b e e n shown to be safe, immunogenic, and phenotypically stable w h e n administered by the intranasal route to healthy adults and children. Two such strains, influenza B/Ann Arb o r / i / 8 6 [22"'] and influenza A/Bethesda/85 (H3N2) [23], have been shown to confer protection against experimental disease in adults and in subjects up to 6 months of age, respectively. Administration of such attenuated vaccines to elderly persons, however, did not elicit a superior local or systemic antibody response in comparison with a killed parenterally administered trivalent vaccine [24]. Large-scale field trials need to be conducted to accurately determine the potential of such attenuated vaccines. While intranasally administered vaccines are well tolerated and would probably increase influenza vaccine usage, the need to generate new vaccine strains on a near-annual basis may prove to be too cumbersome. The OKA live attenuated varicella vaccine strain has b e e n licensed in Japan, Korea, and in several European countries, primarily for use in selected high risk
groups. About 4 % of healthy children and adolescents will experience a varicelliform rash after immunization [25,26], but no serious reactions have been reported. The vaccine provided 95 % protection against disease over two varicella seasons. Children with acute lymphocytic leukemia, which renders them highly susceptible to varicella, were also vaccinated. Varicelliform rashes were more frequent in this group with roughly 5 % of subjects presenting with what appeared to be overt disease. However, this p h e n o m e n o n was associated with steroid therapy just before immunization. The incidence of varicella decreased by 80 % following vaccination.
Conclusion Live attenuated viral and bacterial vaccines have been utilized with great success in the past to control a number of diseases. At present candidates against no fewer than 11 different diseases are undergoing clinical evaluation. Of these the V. cholerae CVD103-HgR vaccine strain is most advanced and is expected to be licensed in the near future. Undoubtedly, others will follow and will significantly expand the number and types of infectious diseases amenable to prevention by vaccination. A highly significant advantage that live attenuated vaccines can provide is the induction of longlasting immunity after only a single dose. This is of critical importance in vaccination campaigns conducted in developing areas where patient access in limited b y logistical considerations.
References and recommended reading Papers of particular interest, p u b l i s h e d within the annual period of review, have b e e n highlighted as: of special interest •. of outstanding interest 1. •.
PROGRAMMEFOR VACCINE DEVELOPMENT (WORLD HEALTH ORGANIZATION):P o t e n t i a l U s e o f Live V i r a l a n d Bacter i a l V e c t o r s f o r V a c c i n e s . V a c c i n e 1990, 8:425-437. A timely review covering the potential of living attenuated viral and bacterial strains to serve as vectors for foreign antigens. Provides a c ompre he ns i ve listing of candidate vectors and their respective adva nt a ge s and drawbacks. 2. Moss B: V a c c i n i a Virus: A T o o l f o r R e s e a r c h a n d Vac•, c i n e D e v e l o p m e n t . S c i e n c e 1991, 252:1662-1667. Presents a c ompre he ns i ve ove rvi e w of the use of vaccinia virus as a n expression vector for delivery of vaccine antigens. 3. •.
COONEY EL, COLLIER AC, GREENBERG PD, COOMBS RW, ZARLINGJ, ARDITrI DE, HOFFMAN MC, HU S-L, COREY L: Safety and Immunological Response to a Recombin a n t V a c c i n i a V i r u s V a c c i n e E x p r e s s i n g HIV E n v e l o p e G l y c o p r o t e i n . L a n c e t 1991, 337:567-572. Illustrates the suppressive effect that prior exposure to or immunization by a vaccine vector may ha ve on the i mmune response to the delivered vaccine antigen. In this instance, a recombinant vaccinia virus expressing gp 160 of h u m a n immunodeficiency virus w as poorl y i mmunoge ni c in subjects w i t h a previous history of vaccination w i t h vaccinia, although it w a s able to evoke good humoral and T-ceU i mmune re s pons e s in naive individuals. 4. •.
STOVEROK, DE LA CRUZ VF, FUERST TR, BIJRLEINJE, BENSON LA, BENNETt LT, BANSAL GP, YOUNG JF, LEE MH, HATFULL
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8.
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16.
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18. •o
SANTOSHAMM, LETSON GW, WOLFF M, REID R, GAHAGAN S, ADAMSR, CALLAHAaNC, SACKRB, KAPIKIANAZ: A Field Study o f t h e S a f e t y a n d Efficacy o f T w o C a n d i d a t e R o t a v i r u s V a c c i n e s i n a Native A m e r i c a n P o p u l a t i o n . J Infect Dis 1991, 163:483-487. Describes a direct comparison of two candidate rotavirus vaccine strains in a blinded, randomized, placebo-controlled trial. Illustrates the fact that efficacy of a given vaccine m a y vary widely d u e to geographical differences and endemic strain. 19.
VESIKARIT, RAUTANENT, VARIS T, BEARDS GM, KAPIKIANAZ: R h e s u s R o t a v i r u s C a n d i d a t e Vaccine: C l i n i c a l T r i a l i n C h i l d r e n V a c c i n a t e d B e t w e e n 2 a n d 5 M o n t h s o f Age. Am J Dis Child 1990, 144:285-289.
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21.
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22. •.
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23.
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S Cryz, Swiss Serum and Vaccine Institute, PO Box 2707, CH-3001 Berne, Switzerland,
Stanley J. Cryz Jr. Swiss Serum and Vaccine Institute, Berne, Switzerland Live attenuated vaccines have been successfully used for the prevention of a number of viral and bacterial diseases. Several vaccine strains have been utilized recently as expression vectors for cloned heterologous antigens. Through the use of recombinant DNA technology, candidate vaccine strains and vector systems have been developed and are undergoing clinical evaluation. Current Opinion in Biotechnology 1992, 3:298-302
Introduction Several live vaccines directed against bacterial and viral pathogens are n o w licensed for h u m a n use (Table 1). The potential of live vaccines is best illustrated by the eradication of smallpox. In several countries, nearly complete immunization against polio, measles, mumps, and rubella has resulted in a dramatic decrease in disease incidence. Eradication of such diseases, where man is the sole natural reservoir, is theoretically possible.
Table 1. Live attenuated vaccines currently licensed for human use.
Disease
Vaccine
Year of introduction
Smallpox Naturally attenuated cowpox virus Tuberculosis Bacille Calmette-Gu~rin (BCG) Yellow fever 17 D strain Polio Trivalent, tissue culture passaged Measles Tissue culture passaged Mumps Tissue culture passaged Rubella Tissue culture passaged Typhoid fever Ty21 a strain Adenovirus Tissue culture passaged Japanese encephalitis Tissue culture passaged Varicella OKA strain, tissue culture passaged
1798 1927 1936 1961 1963 1967 1969 1980 1983 1983 1983
In spite of these successes, infectious diseases still exact a considerable toll worldwide. Vaccines are needed to control parasitic diseases and a number of enteric diseases caused by bacterial and viral pathogens. The application of recombinant DNA technology has led to the construction of numerous novel live vaccine candidates and vectors that hold great promise for the future [1-'].
A number of n e w attenuated vaccine candidates have b e e n developed, some of which are undergoing clinical evaluation (Table 2). This review will primarily focus u p o n the various techniques currently being explored in the development of live attenuated vaccine strains through the use of recombinant DNA technology.
Safety of live attenuated vaccines currently in use
Live vaccines have an outstanding benefit-to-risk ratio and serious post-immunization sequelae are rare. Bacille Calmette-Gu~rin (BCG) tuberculosis vaccine (the most widely used live vaccine with nearly 3 billion doses administered) has a case fatality ratio of roughly 2 per 10 million doses. Approximately 1 in 500 000 recipients of oral polio vaccine will develop poliomyelitis. Aseptic meningitis (non-paralytic polio) lasting 2 to 10 days is a more c o m m o n occurrence but the majority of individuals recover completely. Central nervous system (CNS) disorders following administration of measles vaccine occur at a rate of roughly 1 per 3 million doses. Serious adverse reactions following receipt of rubella or mumps vaccine are rare. These figures demonstrate that severe CNS complaints following immunizatidn with measles, mumps, or rubella vaccine appear at a rate comparable to the background level within the general population. A total of 15 cases of encephalitis has been temporally associated with the administration of 100 million doses of the 17D yellow fever vaccine. Approximately 15 million North American military recruits have received adenovirus vaccine without any substantial untoward effects. Nearly 100 million doses of Ty21a typhoid vaccine have been administered with no serious reaction noted. In summary, live attenuated vaccines rarely cause serious, irreversible, adverse reactions.
Abbreviations BCG--Bacille Calmette-Gu~rin; CMV---cytomegalovirus; CNS~central nervous system; HIV--human immunodeficiency virus; LPS--lipopolysaccharide; RSV--respiratory syncytial virus; TCID--tissue culture infective dose.
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© Current Biology Ltd ISSN 0958-1669
Live attenuated vaccines for human use Cryz 299 I
[Table 2. Live attenuated vaccines under development. Disease
Vaccine
Route
Cholera
A lsubunit deletion mutant Vibrio cholerae CVD103-Hg Salmonella typhi Ty21a expressing V. cholerae O-anti
Oral Oral
Aromatic amino acid (ARO) mutants CRA, CRP deficient mutants Temperature-sensitive mutants
Oral Oral Oral
ARO mutants Escherichia coli-Shigella hybrid strains S. typhi Ty21a expressing 5higella sonnei O-antigen
Oral Oral Oral
E. coli diarrhea
ARO mutants Enterotoxi n-deficient mutants
Oral Oral
Influenza
Cold-adapted virus Avian-human reassortant virus Cold-adapted reassortant virus
Intranasal Intranasal Intranasal
Rotavirus
Rhesus rotavirus, attenuated by tissue culture passage Bovine rotavirus, attenuated by tissue culture passage Human rotavirus, attenuated by tissue culture passage
Oral Oral Oral
Hepatitis A
Attenuated by tissue culture passage
Parenteral
Cytomegalovirus
Attenuated by tissue culture passage
Parenteral
Dengue
Attenuated by tissue culture passage
Parenteral
Acquired immunodeficiency syndrome
Vaccinia virus expressing HIV antigens Sabin type 1 polio virus expressing HIV antigens BCG expressing HIV antigens
Parenteral Oral Parenteral or oral
Hepatitis B
Adenovirus expressing hepatitis B surface antigen
Parenteral
Typhoid fever
Shigellosis
Approaches to live vaccine development
tors, holds great promise for the construction of multivalent live vaccines [1"]. Alternatively, strains can be attenuated by the deletion or inactivation of genes with k n o w n virulence-enhancing attributes. Several such recombinant strains have been developed and are undergoing clinical evaluation.
With one exception, the Salmonella typhi Ty21a strain, all live vaccines currently in use were developed empirically. Attenuation of viral vaccines was accomplished by repeated passaging on tissue culture ceils, eggs, or a combination of the two. Several passage levels were then selected and tested in an effort to isolate an avirulent virus capable of sufficient replication in man to engender a protective immune response without attendant adverse reactions. The BCG vaccine strain was attenuated by serial passage of Mycobacterium bovis for 13 years. The two major drawbacks to such an approach are undefined attenuating mutation(s) and potential strain instability. Even so, these vaccines have been proven to be remarkably safe and effective. This approach continues to be used today in the development of new vaccine strains.
The use of live vaccine strains as vectors for candidate vaccine antigens is particularly attractive for several reasons: their safety has already been established; they have the potential to immunize against two or more disease entities; and recombinant strains hyper-producing one or more protective antigens might prove to be more effective as immunizing agents than the attenuated pathogen itself.
The application of recombinant DNA technology to live vaccine development has allowed for a more rational approach. The identification of critical protective antigens, followed by cloning and expression in live vec-
A number of foreign genes have been inserted into the smallpox vaccine (Vaccinia virus) [1"'-3"]. Several such recombinant strains have been shown to afford protection against bacterial and viral pathogens in ex-
Live vaccines based upon vector systems
300 Evironmentalbiotechnology perimental systems. O n e drawback to this approach in humans is the unacceptably high rate of reactions associated with the smallpox vaccine. To circumvent this problem, several genes have b e e n deleted to further attenuate this strain. A thymidine kinase-deficient variant expressing h u m a n immunodeficiency virus (HIV) gp 160 w a s found to be safe in HIV seronegative males [3"']. However, in subjects w h o had previously b e e n immunized against smallpox it engendered only a weak, transient anti-HIV immune response. In contrast, subjects w h o had not previously b e e n immunized m o u n t e d a vigorous, long lasting anti-HIV T-cell and antibody response. These results suggest that preexisting immunity to a vaccine vector m a y suppress the host's ability to respond to the recombinant antigen. The use of the BCG vaccine strain as a vector has m a n y potential advantages including its d o c u m e n t e d safety, low cost, and current widespread use as a childhood vaccine, as well as the ability of a single dose of the vaccine to induce long-lasting protection. Several recombinant BCG strains have b e e n constructed that stably express foreign genes, including several from IMV [4"',5]. Preliminary results in animals have demonstrated that these constructs can induce both humoral and cell-mediated i m m u n e responses. Genes encoding antigens from h u m a n papilloma virus, hepatitis A virus, influenza virus, Herpes simplex virus, HIV, respiratory syncytial virus (RSV), and coxsackie B virus have b e e n inserted into the g e n o m e of Sabin type 1 oral polio virus [1"']. Several of these recombinant strains have b e e n s h o w n to induce a neutralizing antibody response to both polio virus and the heterologous pathogen. However, in several instances, antibody was unable to recognize the native antigen as expressed b y the heterologous pathogen. Recombinant adenovirus type 4 and 7 strains expressing hepatitis B surface antigen have b e e n constructed [6]. Chimpanzees immunized by the oral route with these strains were protected against hepatitis B challenge. Several HIV genes and the gene encoding the F glycoprotein of RSV have also b e e n introduced into adenovirus [1"]. Immunization of cotton rats with the F glycoprotein recombinant protected against RSV challenge. As the S. typhi Ty21a strain has a remarkable safety record and proven efficacy [7"], it has b e e n utilized as a vector expressing the antigens of other enteric pathogens. The rfb genes of Shigella sonnei and Vibrio cholerae, which code for the O-antigen moiety of lipopolysaccharide (LPS), have b e e n introduced into Ty21a [8,9]. The S. typhi-S, sonnei hybrid provided a modest degree of protection in volunteers against a S. sonnei challenge [8]. However, efficacy varied from one batch to another.':Although the precise reason for this p h e n o m e n o n remains unknown, it m a y be related in part to the in vivo instability of the plasmid coding for the O-antigen, or to the fact that the O-antigen is not covalently linked to the core of S. typhi LPS. The S. typhi-V, cholerae hybrid contains the rfa gene encoding Escherichia coli LPS core and the rfb gene of V. cholerae, which allows for the attachment of the V.
cholerae O-antigen to the E. coli core. Multiple doses of this vaccine resulted in a 25 % reduction in the diarrhea attack rate following a V. cholerae challenge [9]. This modest protection was attributed to the low levels of antibody to V. cholerae O-antigen induced by the vaccine. Inactivation of the S. typhi u~b gene, which allows for m o r e efficient expression of the V. cholerae O-antigen on the cell surface, significantly increased the immune response in humans [10]. The efficacy of this strain is currently under evaluation.
Live candidate vaccines developed through recombinant DNA technology A live, oral, attenuated cholera vaccine termed CVD103-HgR has been developed b y deletion of the enzymatically active A 1 subunit of cholera toxin [11]. This strain has b e e n found to be safe and i m m u n o g e n i c in double-blind, randomized, placebo-controlled trials in North American adults, Swiss adults [12], Thai adults [13], and in Indonesian children between 2 and 9 years of age (MM Levine, personnal communication). A single orally administered dose of vaccine provided high levels of protection (62-100%) against experimental challenge with V. cholerae, which last for at least 6 months [11] (M Levine, unpublished data). E x p a n d e d Phase II safety and immunogenicity studies are n o w u n d e r w a y in Asia and South America as a prelude to field trials in cholera-endemic areas. Bacterial auxotrophic mutants with complete blocks in aromatic amino acid synthesis pathway (ARO) are avirulent in mammalian hosts because of their inability to produce or acquire p-aminobenzoic acid (a folic acid precursor) and 2,3-dihydroxybenzoic acid (a precursor of bacterial siderophores) from the host [14"]. A Shigellaflexneri aroD deletion mutant has b e e n found to be safe, immunogenic, and protective in m o n k e y studies [14"]. H o n e et al. [15] have constructed double aro mutants b y deletion of the aroC and D genes in S. typhi. One such strain, CVD908, has b e e n f o u n d to be safe and remarkably immunogenic in humans after a single oral dose (M Levine, personal c o m m u nication). ARO mutants m a y prove to be highly effective live oral vaccines against several enteric diseases because of their safety and apparent undiminished immunogenicity. These traits could also make t h e m ideal vectors for the delivery of heterologous antigens.
Live candidate vaccines developed by classical methods of attenuation T w o live strains of hepatitis A, attenuated b y passage on cultured cells, are currently undergoing clinical evaluation [16,17]. The H2 strain has b e e n parenterally administered to 12 healthy seronegative adults and 127 children, 4-12 years old at a tissue culture infective dose (TCID50) of 106.5 [16]. There was n o evidence of liver toxicity following vaccination and all subjects seroconverted. One potential drawback of this vaccine is that it is cultured on the KMB17 h u m a n lung fibroblast cell line, a substrate not routinely used for propaga-
Live attenuated vaccinesfor human use Cryz 301 tion of human vaccines. The F' variant of strain CR326F was also well tolerated w h e n parenterally administered to volunteers [17]. At a dose of 107.3 TCID50 , 10 out of 10 subjects seroconverted. Both vaccines induced longlived humoral immune responses and neutralizing antibodies to hepatitis A vires (assayed in vitro). Although such live attenuated vaccines were less immunogenic than inactivated hepatitis A vaccines administered by a multidose regimen, they may prove to be highly effective as a result of efficient priming of the immune system and the need for only a single dose. Several live attenuated rotavirus candidate vaccine strains have been developed and field tested [18"]. Results from Finnish and South American studies have shown highly significant protection. The same strains, however, were found to be ineffective when tested in other geographical areas [18"',19]. In some cases, lack of protection could be explained by the fact that the endemic field strains were of a heterologous serotype. In other trials, differences in efficacy could be related to the age of immunization. Protection was lower in infants, w h o generally mounted a poorer immune response. It n o w appears that a multi-serotype rotavims vaccine that is highly immunogenic in infants will be needed. The Towne live attenuated strain of cytomegalovirus (CMV) was derived after 105 passages o n h u m a n diploid ceils. Immunization of seronegative subjects resulted in partial protection following parenteral challenge with a low passage strain of CMV [20]. Naturally seropositive subjects were resistant to a 10-fold higher challenge dose of vires than the Towne strain recipients. Immunization of seronegative renal transplant recipients, w h o subsequently received an organ from a seropositive donor, resulted in a significant decrease in disease scores compared with a placebo group [21]. Graft survival was also higher in the vaccinated group. Immunization did not, however, influence the rate of infection. Several live-attenuated, cold-adapted reassortant influenza vaccine strains have b e e n shown to be safe, immunogenic, and phenotypically stable w h e n administered by the intranasal route to healthy adults and children. Two such strains, influenza B/Ann Arb o r / i / 8 6 [22"'] and influenza A/Bethesda/85 (H3N2) [23], have been shown to confer protection against experimental disease in adults and in subjects up to 6 months of age, respectively. Administration of such attenuated vaccines to elderly persons, however, did not elicit a superior local or systemic antibody response in comparison with a killed parenterally administered trivalent vaccine [24]. Large-scale field trials need to be conducted to accurately determine the potential of such attenuated vaccines. While intranasally administered vaccines are well tolerated and would probably increase influenza vaccine usage, the need to generate new vaccine strains on a near-annual basis may prove to be too cumbersome. The OKA live attenuated varicella vaccine strain has b e e n licensed in Japan, Korea, and in several European countries, primarily for use in selected high risk
groups. About 4 % of healthy children and adolescents will experience a varicelliform rash after immunization [25,26], but no serious reactions have been reported. The vaccine provided 95 % protection against disease over two varicella seasons. Children with acute lymphocytic leukemia, which renders them highly susceptible to varicella, were also vaccinated. Varicelliform rashes were more frequent in this group with roughly 5 % of subjects presenting with what appeared to be overt disease. However, this p h e n o m e n o n was associated with steroid therapy just before immunization. The incidence of varicella decreased by 80 % following vaccination.
Conclusion Live attenuated viral and bacterial vaccines have been utilized with great success in the past to control a number of diseases. At present candidates against no fewer than 11 different diseases are undergoing clinical evaluation. Of these the V. cholerae CVD103-HgR vaccine strain is most advanced and is expected to be licensed in the near future. Undoubtedly, others will follow and will significantly expand the number and types of infectious diseases amenable to prevention by vaccination. A highly significant advantage that live attenuated vaccines can provide is the induction of longlasting immunity after only a single dose. This is of critical importance in vaccination campaigns conducted in developing areas where patient access in limited b y logistical considerations.
References and recommended reading Papers of particular interest, p u b l i s h e d within the annual period of review, have b e e n highlighted as: of special interest •. of outstanding interest 1. •.
PROGRAMMEFOR VACCINE DEVELOPMENT (WORLD HEALTH ORGANIZATION):P o t e n t i a l U s e o f Live V i r a l a n d Bacter i a l V e c t o r s f o r V a c c i n e s . V a c c i n e 1990, 8:425-437. A timely review covering the potential of living attenuated viral and bacterial strains to serve as vectors for foreign antigens. Provides a c ompre he ns i ve listing of candidate vectors and their respective adva nt a ge s and drawbacks. 2. Moss B: V a c c i n i a Virus: A T o o l f o r R e s e a r c h a n d Vac•, c i n e D e v e l o p m e n t . S c i e n c e 1991, 252:1662-1667. Presents a c ompre he ns i ve ove rvi e w of the use of vaccinia virus as a n expression vector for delivery of vaccine antigens. 3. •.
COONEY EL, COLLIER AC, GREENBERG PD, COOMBS RW, ZARLINGJ, ARDITrI DE, HOFFMAN MC, HU S-L, COREY L: Safety and Immunological Response to a Recombin a n t V a c c i n i a V i r u s V a c c i n e E x p r e s s i n g HIV E n v e l o p e G l y c o p r o t e i n . L a n c e t 1991, 337:567-572. Illustrates the suppressive effect that prior exposure to or immunization by a vaccine vector may ha ve on the i mmune response to the delivered vaccine antigen. In this instance, a recombinant vaccinia virus expressing gp 160 of h u m a n immunodeficiency virus w as poorl y i mmunoge ni c in subjects w i t h a previous history of vaccination w i t h vaccinia, although it w a s able to evoke good humoral and T-ceU i mmune re s pons e s in naive individuals. 4. •.
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ALDOVINIA, YOUNG RA: H u m o r a l a n d C e l l - m e d i a t e d Imm u n e R e s p o n s e s to Live R e c o m b i n a n t BCG*HIV Vacc i n e s . Nature 1991, 351:479-482.
6.
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SIMANJUNTAK CH, PALEOLOGOFP, PUNJABINH, DARMOWIGOTO R, SOEPRAWOTO, TOTOSUD1RJO H, HARYANTO P, SUPRIJANTO E, WITHAM ND, HOFFMAN SL: O r a l l r n r n u n i s a t i o n A g a i n s t T y p h o i d Fever i n I n d o n e s i a w i t h Ty21a V a c c i n e . Lancet 1991, 338:1055-1059. This study demonstrated the ability of the Ty21a live oral vaccine to provide highly significant protection against disease in a hyperendemic area. The authors conclude that routine immunization would provide substantial public health benefits.
8.
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16.
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17.
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18. •o
SANTOSHAMM, LETSON GW, WOLFF M, REID R, GAHAGAN S, ADAMSR, CALLAHAaNC, SACKRB, KAPIKIANAZ: A Field Study o f t h e S a f e t y a n d Efficacy o f T w o C a n d i d a t e R o t a v i r u s V a c c i n e s i n a Native A m e r i c a n P o p u l a t i o n . J Infect Dis 1991, 163:483-487. Describes a direct comparison of two candidate rotavirus vaccine strains in a blinded, randomized, placebo-controlled trial. Illustrates the fact that efficacy of a given vaccine m a y vary widely d u e to geographical differences and endemic strain. 19.
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20.
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21.
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22. •.
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23.
STEINHOFF Me, HALSEY NA, WILSON MH, BURNS BA, SAMORODIN RK, FRIES LF, MURPHY BR, CLEMENTS ML: C o m p a r i s o n o f Live Attenuated Cold-adapted a n d A v i a n H u m a n InfluenZa A / B e t h e s d a / 8 5 (H3N2) Reassortant V i r u s V a c c i n e s i n I n f a n t s a n d C h i l d r e n . J Infect Dis 1990, 162:394-401.
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24.
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POWERSDC, FRIES LF, MURPHY BR, THUMARB, CLEMENTSME: I n Elderly Persons Live Attenuated I n f l u e n z a A V i r u s Vaccines do not Offer an Advantage over Inactivated V i r u s V a c c i n e i n I n d u c i n g S e r u m or S e c r e t o r y Antib o d i e s o r Local I m r m m o l o g i c M e m o r y . J Clin Microbiol 1991, 29:498-505. 4
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14. •,
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S Cryz, Swiss Serum and Vaccine Institute, PO Box 2707, CH-3001 Berne, Switzerland,