- Email: [email protected]
Recombinant vaccine delivery systems and encoded vaccines
Recombinant vaccine delivery systems and encoded vaccines C Kendall Stover PathoGenesis
Corporation,
Seattle, USA
In addition to the introductory demonstrations of genetic immunization, the past year has brought significant advances in vaccine development. Particularly encouraging are live recombinant vaccines, studies demonstrating the potential to elicit both systemic and mucosal immune responses, more studies demonstrating immune protection in animal disease models, and experimental
vaccines eliciting
immune
Current Opinion in Immunology
responses
1994,
in primates.
6:568-571
Introduction
live recombinant bacterial vaccine vehicles
Despite an ever increasing knowledge of immune mechanisms and protective antigens, the principal obstacles impeding the development of use&l and sail subunit vaccines have not changed significantly over the last 5 years. The enormous task of cost-effectively producing sufficient quantities of a potentially protective imrnunogen is one obstacle that often kills vaccine development efforts. The problems inherent in providing the immunogen in a safe format that still promotes the desired protective immune response are often insurmountable. Most vaccine delivery systems aim to minimize the impact of these major obstacles. Vaccine delivery systems can be roughly categorized as particulate vaccine carriers, live vaccine vehicles, and encoded vaccines. Particulate vaccine carriers (e.g. virus-like particles, liposomes and immune-stimulating complexes or ISCOMs) aim to enhance irmnunogenicity to such an extent that less of the immunogen is necessary to achieve the desired protective response. Such systems will not be covered in this review. Live vaccine vehicles, for example bacille calmette-guerin (BCG), Salmonella and pox viruses preclude the need to produce and purifjr protective immunogens, as these are expressed by the vehicle in the vaccinee upon introduction in viuo. The most recent strategy of genetic immunization employs encoded vaccines composed of the DNA or RNA of an immunogen. These encoded vaccines are directly introduced or transfected into tissues which then express the desired immunogen &om the encoded vaccine template, thereby obviating the need to design production and formulation processes for each immunogen. The past year has brought fLrther incremental advances in the understanding, and use, of live vaccine vehicles and the initial demonstrations of genetic immunization.
Among the bacteria being considered for live recombinant vaccine vehicles (LRWs), the most prominent are still attenuated Salmonella and mycobacterial BCG. While, previous studies have demonstrated protective responses to recombinant antigens expressed in attenuated Salmonella, the past year has brought the first reports of protective responses to recombinant antigens expressed in the recombinant BCG (rBCG) vaccine vehicle. A systematic comparison of antigen expression by rBCG within the cytoplasm, in the membrane as a chimeric lipoprotein, or as a secreted and cell wall associated protein, resulted in the construction of a promising vaccine against Lyme disease [ 1.1. Potent and lasting antibody responses were elicited to the OspA antigen of Ebwelia burgdotfeti, when it was expressed as a chimeric lipoprotein in the membrane of rBCG. These responses compared favorably with subunit vaccines based on OspA lipoprotein and are particularly noteworthy because it was anticipated that rBCG-based vaccines would be most suitable for eliciting cellular responses. It is likely that an rBCG-OspA vaccine will be the first one to be tested in humans. Also of note is an rBCG vaccine expressing a surf&e protein, PspA, of Streptococcus pneumoniae. Mice immunized with an rBCG-PspA vaccine showed protective humoral responses against virulent Pneumococcus and rBCG-PspA vaccines effectively primed with purified PspA-subunit vaccines (S Langermann, MS Hanson, JE Burlein, SR Palaszynski, LS McDaniel, DE Briles, CK Stover, unpublished data). A recent study with an rBCG vaccine expressing the gp63 antigen of Leishmaniu also indicates that protective cellular responses, which rBCG vaccines were originally intended for, can also be elicited to recombinant antigens expressed by rBCG [2].
Abbreviations BCG-bacille calmette-guerin; CTL-cytotoxic T lymphocyte; ISCOM-immune-stimulating MHC-major
568
complex; LRW-live recombinant vaccine vehicle; LT-B-heat-labile enterotoxin; histocompatibility complex; rBCC_recombinant BCG; W-simian immunodeficiency virus.
0 Current Biology Ltd ISSN 0952-7915
Recombinant vaccine deliverv svstems and encoded vaccines Stover
The first published studies with an rBCG vaccine in primates demonstrated specific MHC-restricted CD8+ cytotoxic T lymphocyte (CTL) responses to the Simian immunodeficiency virus (SIV)-mat gag epitope [3]. This study confirmed earlier findings [4] indicating a potential for rBCG to elicit class I restricted cellular responses even though BCG persists in the endosomal phagolysosome of macrophages and not in the cytoplasm where class I antigen presentation pathway is thought to operate. The first demonstration for the potential of rBCG to elicit antigen-specific mucosal responses was also reported in the past year (S Langermann, S Palaszynski, A Sadziene, S Koenig, CK Stover, unpublished data; [5]). A single intranasal immunization with rBCG-OspA or rBCG-PspA elicited substantial mucosal and systemic immune responses comparable to parental immunization with the same rBCG vaccines. This finding is particularly significant in comparison with Salmonella, which, as an enteric bacteria, is considered to be most promising for use as an oral vaccine for eliciting mucosal responses. Although BCG is still one of the most widely used vaccines in the world, one of the disadvantages of using rBCG vaccine is the controversy surrounding its efficacy against tuberculosis. Therefore, one of the most significant studies affecting potential rBCG vaccines is a retrospective statistical study that found the BCG tuberculosis vaccine to be significantly more effective than widely believed, particularly against serious forms of tuberculosis and mortality [6*]. The past year also brought incremental advances in the understanding and development of Salmonella as a live vaccine vehicle. To further enhance Sulmonella’s ability to elicit mucosal responses by oral immunization, expression vectors have been designed that permit easy &sion to heat-labile enterotoxin (LT-B) of Escheridh coli which take advantage of the mucosal imrnunostimulatory properties of LT-B [7]. These vectors allow fusions via a linker to the carboxy-terminal end of LT-B resulting in a pentameric LT-B antigen f&ion protein which should retain LT-B GM1 ganglioside-binding activity. Further study with LT-B antigen fusions in recombinant Salmonella vaccine vehicles is necessary to determine whether this combination of vaccine delivery strategies offers real advantages over each strategy alone. In a surprising study, Salmonellavaccines expressing higher levels of antigen horn an unstable plasmid vector, which was lost within 24 h of immunization, resulted in higher levels of antigen specific antibody (systemic and mucosal) than Salmonella expressing lower levels of antigen horn a much more stable (at least 21 days) single copy chromosomal vector [&I. This study suggests that the most important event in the development of an immune response against a foreign antigen delivered by Salmonella (and possibly other bacterial vector systems) may be the initial amount of antigen priming the immune response and not the persistence of the bacterial vector in tissues. This is somewhat surprising as considerable effort has been spent in developing stable vector systems for Salmonella and BCG to ensure longer term persistence of the bacterial vehicle.
live recombinant viral vaccine vehicles The most widely studied live vaccine vehicles are recombinant vaccinia viral vectors. Further progress was demonstrated in the potential for vaccinia to elicit substantial and relevant immune responses, and the potential for using recombinant vaccinia virus HIV-l antigens to prime for immunization with subunit vaccines or whole inactivated virus vaccines was reported in primate studies [9,10]. A study evaluating the effect of two different recombinant vaccinia virus immunizations on a single host revealed that priming with the first vaccinia virus can inhibit the titer and duration of an antibody response elicited by a second distinct recombinant vaccinia virus for nine months in mice [l 11. While vaccinia-based veterinary vaccines are being pursued, concerns about the safe use of recombinant vaccinia vectors have resulted in the engineering of the highly attenuated NYVAC vaccinia virus and the canary pox ALVAC virus for use as live vaccine vehicles [12,13]. Despite the drastic attenuation of NYVAC and the inability of the ALVAC virus to replicate in mammalian cells, evidence is mounting that these recombinant viruses can elicit strong and protective immune responses to extrinsic antigens expressed in these systems [14-161. Studies on the use of recombinant adenovirus as live viral vaccines proliferated in 1993. Recombinant adenovirus is thought to be particularly promising for generating mucosal immune responses. Promising systemic and mucosal immune responses to rotovirus VP7 and herpes simplex virus glycoprotein B antigens were demonstrated in two different studies using intranasal immunization with recombinant adenovirus [17,18]. In chimpanzees, recombinant human adenovirus expressing HIV-l env or gag-protease genes were used in multiple route inoculation regimens, which included boosting with subunit vaccines [19]. A variety of cellular and humoral responses were observed in this study including low-titered type-specific neutralizing antibodies and mucosal HIV-specific antibodies. New approaches to engineer hybrid live viruses and chimeric non-replicating pseudovirions have also been reported recently [20*,21]. It is likely that such approaches will proliferate in the near titure as the mechanisms of viral assembly for a variety of viruses are better understood.
Encoded vaccines and genetic immunization Perhaps the most noteworthy and controversial event in vaccine development in the past year was the first report of immune responses to genetic immunization using encoded vaccines. With this strategy of immunization, a genetic template (DNA or RNA) is directly injected into tissues, resulting in transfection of host cells and endogenous expression of the antigen encoded by the genetic template (encoded vaccine). This strategy eliminates the need to design specific processes for purification and formulation of different antigens and only requires the simpler and relatively standard method for purification of nucleic acid. A number of genetic
569
570
Immunity to infection
immunization studies employing DNA encoding viral antigens or HIV-l or influenza viruses by intramuscular injection have demonstrated antigen-specific cellular and humoral immune responses .that were protective in mice and chickens or contained HIV-l viral neutralizing activity [22,23”,24]. Some of these studies indicate that DNA genetic immunization can be accomplished fairly efficiently by multiple routes of inoculation including mucosal routes [25*,26]. Another strategy of genetic immunization employed injection of liposome entrapped mRNA encoding influenza virus nucleoprotein (NP) [27]. In this study, MHC-restricted CTL responses to NP were elicited in mice of three different haplotypes. These CTL responses were very similar to those elicited by the live influenza virus in the same mouse strains. To elicit these responses, the mRNA encoding the NP antigen had to be encapsulated presumably because of m.RNA lability. While the preparation of mRNA and its encapsulation into liposomes adds two levels of complexity to the use of DNA for genetic immunization, the use of mRNA greatly reduces the potential risk of mutations resulting fi-om integration of viral DNA into the host genome. While these first genetic immunization studies have demonstrated the potential for encoded vaccines to elicit both cellular and humoral immune responses, f%rther characterization of such immune responses and comparisons with other vaccine delivery systems are necessary to evaluate the real potential for encoded vaccines.
Conclusions There are two basic problems confronting vaccine development: cost-effective production and purification of antigens f?om a pathogen or expression system in sufflcient quantities for vaccine studies; and the delivery of this antigen in a suitable and safe format (adjuvant or carrier) to promote protective immune responses. LRWs offer significant advantages over subunit vaccines because antigen immunogen purification is eliminated and the source of recombinant antigen replicates in the host, potentially providing a means of auto-boosting. An organism producing antigen is thought to be innately more immunogenic as the antigen is more likely to be recognized as foreign and processed by antigen-presenting cells. A variety of attenuated bacteria and viruses are currently under investigation as live vaccine vehicles. Despite the obvious advantages that LRWs potentially offer, they are generally not as palatable as subunit vaccines, especially in the USA, where no current live attenuated vaccines are used. Some of the reasons for this negative perception of LRWs may be more intuitive than factbased. It is a f&t that all humans carry commensal live replicating organisms, some of which are very beneficial. It also seems that many percieve the LRWs to be only a temporary solution or an inexpensive way to make vaccines for the developing world. While there are well founded concerns about the use of LRWs, particularly in immunosuppressed populations, we must also consider the problems associated with formulating safe immuno-
genic subunit vaccines and practical considerations, such as cost. Encoded vaccines seem to offer some of the advantages of a live vaccine without the organism vehicle. Concerns about eliciting anti-nucleic acid immune responses and the possibility that injecting large amounts of genetic material could cause mutations following insertional events in the host genome may be d&cult to overcome despite the available technology. As always, the benefits of new technologies must be weighed against risks which are both real and documented or perceived risks which may only be speculative. Some of the most significant progress to come in the next few years may be the availability of new technologies to deal with safety issues, however such advances usually lag behind, as in the case of pox virus delivery systems and the recent development of safer NYVAC and ALVAC viruses. The very latest developments in vaccine technology are reviewed in [28’,29*,30].Developments in gene therapy and approval of protocols for human trials may help pave the way for genetic immunization in humans. It is likely that we will see human clinical trials with some of the newer LRWs and encoded vaccine technologies in the next few years.
Acknowledgements I appreciate the helpful discussions on this topic with Dt TR Fuent of Genelabs Technologies, Dr S Langerman of MedImmune Incorporated, and Dr M Collett of PathoGenesis Corporation, and the assistance of S Colwell in the preparation of this manuscript.
References and recommended reading Papers of particular interest, published within the annual review, have been highlighted . of special interest .. of outstanding interest
period of
as:
1. .
Stover CK, Bansai GP, Hanson MS, Buriein JE, Palaszynski SR, Young IF, Koenig 5, Young DB, Sadziene A, Barbour AC: Prohitd by .Recombinani Bacille Calmettetect&~ immky Cuerin @CC) Ewin~~ Outer Surface Protein A (*A) lipoprotein: A Cadit; Lyme Disease Vaccine. J Exp A&d 1993, 178:197-209. A systematic comparison of antibody responses lo recombinant antigen (CkpA) expressed within the cytoplasm, secreted or in the membrane of rBCC as a chimeric lipoprotein. This study resulted in the construction of a promising live rBCC vaccine against Lyme disease. Strong, longlasting protective antibody responses were elicited to OspA expressed as a chimeric lipoprotein in the membrane of rBCG. These responses compare very favorably with subunit vaccines based on OspA lipoprotein and are particularly noteworthy because it was anticipated that rBCCbased vaccines would be most suitable for eliciting cellular responses. 2.
Conneil ND, Medina-Acosta E, Mcmaster WR, Bloom BR, Russell DC: Effective ImmunizationagainstCutanious Leishmaniaxis with Recombinant Bacilk CabneltcGuerin Expressing the Leishmanis Surface Pmteinase gp63. Proc Nati Acad Sci USA 1993, 90:11473-l 1437.
3.
Yasutorni YS, Koenig S, Haun SC, Stover CK, Emini E, Fuerst TR, RCG-SW &its Le?vin NL: Itnmum ‘zation with Recombkant SW-Specific Cytotoxic T Lpqhocytes in Rhesus Monkeys. / lmmonol 1993, 150:3101-3107.
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Stover CK, De La Cruz V, Burlein JE, Lee MH, Hatfull GF, Young JF, Snapper SB, Jacobs WR, Fuerst TR, Bloom BA: New Use of BCC for Recombinant Vaccines. Nature 1991, 351:456-460.
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Lagranderie M, Murray A, Cicquel B, Lecierc C, Gheotghiu M: Oral Immunization with Recombinant BCC Induces Cellular
bom Mice from Naive Dams Vaccinated with a Single Dose of a Recombinant Adenovirus ExpressingRotavirus VP7sc. viro/ogy 1993, 193:94&950.
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TF, Berkey CS, Wilson ME, Burdick E, Finebera HV, Mosteller F: Efficacy of BCC Vaccine in the Pre. vent%n”of kbefcubsis. j Am M&d Assoc 1994, 271:698-702. This study is a statistical meta-analysis of published clinical trials that BCC is 50% efficacious in preventing tuberculosis and 70-80% efficacious in preventing tuberculosis meningitis and death from tuberculosis. These findings are particularly significant because one of the major negatives aspects of the new rBCG vaccines is the controversy surrounding BCG’s efficacy against tuberculosis and its certain perturbation of surveillance procedures in the USA. In light of the TB resurgence, this study suggests that BCC immunization should be reconsidered.
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;;l~icity of Recumbiynt_Huny McnovinwHuman nwnoddctency Virus Vaccmesm Chimpanzees. AIDS Res Hum Refroviruses
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Cardenas L, Clements JO: Stability, lmmunogenicity and Expression of Foreign Antigens in Bacterial Vaccine Vectors. Vaccine
1993, 11:126-135. A surprising study, suggesting that the most important event in the development of an immune response against a foreign antigen delivered by Salmonella, (and possibly other bacterial vector systems) may be the initial amount of antigen priming the immune response and not the persistence of the bacterial vector in tissues.
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Cooney EL, Mcelrath MJ, Corey L, Hu SL, Collier AC, Arditti D, Hoffman M, Coombs RW, Smith GE, Greenberg PD: Enhanced
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Wang B, Ugen KE, Srikantan V, Agadjanyan MC, Dang K, Refaeli Y, Sato Al, Boyer J, Williams WV, Weiner DB: Gene In-
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A: Heter&gous Protection against Influenza by Injection of DNA Encoding a viral Protein. Science 1993, 259~1745-1749.
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Tartaglia J, Cox WI, Taylor I, Perkus M, Riviere M, Meignier B, Paoletti E: Hifly Attenuated Poxvirus Vectors. AIDS Res Hum Retroviruses 1992, 8:1445-l 447.
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Tartaglia J, Perkus ME, Taylor J, Norton EK, Audonnet JC, Cox WI, Davis SW, Van Der Hoeven J, Meignier 8, Riviere M, et a/.: NWAC: a H*ly Attenuated Strain of Vaccinia Virus. tiro/ogy 1992, 18&217-232.
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J, _Paoletti E: induction of Cytotoxic T LymRccombuunt Canarypox (ALVAC) and Attenuated kacchia (fiWAC) Viruses Expre&ngthe HIV-1 Envelope Clycoproteio. virology 1993, 195:845-850. P, Paoletti E: ProFelii leukemia Virus by Vaccination with a Canarypox Virus Recombinant, ALVAC-FL. / \/irol 1993,
Tartaglia J, Jarrett 0,
tectionof Cats a@st
Neil JC, Desmettre
Konishi E, Pincus S, Paoletti E, Laegreid WW, Shape RE, Mason pw: A Highly Attenuated Host Range-Restricted Vaccinia
Virus St& NWAC, Encodii the prhi, E, and NSI Genes of JapaneseEncephalRis Virus Prevents JEV Viremia in Swine. tirchgy 1992, 19Bz454-458. Gallichan WS,
Johnson
DC, Graham FL,
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KL:
Fynan EF, Robinson HL, Webster RG: Use of DNA encoding influenza hemaggluthii as an avian influenza vaccine. DNA Cell Biol 1993, 12:785-789.
Fynan EF, Webster RG, Fuller DH, Haynes JR, Santoro JC, Robinson HL: DNA Vaccines; Protective Immunizations by Parenteral, Mucosal, and GCM-Gun Inoculations. Proc Nat/ Acad Sci USA 1993, 90~11478-11482. This study indicates that DNA vaccine immunization has the potential to be used to immunize by multiple routes and to elicit both systemic and mucosal responses with practice amounts of DNA and immunization schemes.
25. .
26.
Robinson HL, Hunt LA, Webster RG: Protection agilinst a Lethal tnfhnza Virus Challenge by lmmuoixatii with a Haemagglutinin-Expressing Plasmid DNA. Vaccine 1993, 11:957-960.
27.
Mattinon F, Krishnan S, Lenren G, Magno R, Gomard E, Guillet J-G, Lovy J-P, Meulien P: Induction of ViNS-specific cytotoxic T Lymphocytes In viva by liposomc-entrapped mRNA. Eur J lmmunol 1993, 23:1719-1722.
~~i,~yrtaglia
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90~4156-4160.
23.
90:1882-l
11.
16.
oculation Generates Immune Respomes Against Human Immunodeficiency Virus Type 1. Proc Nat/ Acad Sci USA 1993,
In this study CTLs to conserved regions of influenza A virus NP were elicited by intramuscular injection of DNA encoding NP. These responses resulted in some protection against challenge with a heterologous influenza A virus in this influenza mouse challenge model.
1994,
15.
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Brown CS. Welling-Webster S. Feulbrief M. Van Lent JWM. . Spaan WJM: Ch&ic l?aw&us 819 Cap&es for the Presentation of For&n Eoitooes. virolow 1994, 477~477-488. A nice study in which chit&ri;pa&virus Blycapsids were constructed to canv linear eoitooes form human heroes simplex virus type 1 (HSV) and m&se hepaiitis’virus A59 (MHV). These ep&pes were’exposed on the surface of the chimeric 819 capsids and elicited a partially protective response to lethal challenge with either MHV or HSV.
hmunity to Human lmmunodeficiency Virus?HIV) Envelope Elicited by a Combined Vaccine Regimen Consistingof Priming with a Vaccinii Recombinant Expreshg HIV Envelop? and Boosting with gp16B Protein. Proc Narl Acad Sci USA 1993,
complete Protection, but Suppression of Virus Burden, Elicited by SubuoR Simian lnnnudcf~iency Virus Vaccines. J Wrol
14.
1993,
20.
61:1&4-1015.
.
Natuk RJ, Lubeck MD, Chanda PK, Chengalvala M, Wade MS, Murthy SC, Wilhelm J, Vernon SK, Dheer SK, Mizutani
Edelman R: Vaccine% New Technologies and Applications. Vaccine 1993, 11:1361-1364. A nice shalt review of all the vaccine technologies discussed at a vaccine meeting held March 22-24 of 1993. 28.
.
Ginsbere HS, Brown F, Chanok RM, Lemer RA: Vaccines93: Modem”Appioaches To New Vaccines including Prevention Of A/US. Cold Spring. Harbor: Cold Spring_ Harbor Laboraton/ Press; 1993. . This compendium of papers from the Cold Spring Harbor Vaccines ‘93 meeting includes much of the vaccine delivery systems technology currently being evaluated. 29.
.
30.
Mu-
Brown F:Recombinant Vectors in Vaccine _t. Dev Bio/ Stand, vol 82. Basel: Karger; 1994.
In
cosal Immunity and Protection after Intranasal Immunization with Recombinant Adenvirus ExpressingHerpes Simplex virus Glycoprotein B. ) Infect Dis 1993, 168z622-629. 18.
CK
Stover,
Both GW, Lockett LJ, Janardhana V, Edwards SJ, Bellamy AR, Graham FL, Prevec L, Andrew ME: Protective immunity to
PathoGenesis
Rotaviru+lnduced Diirrhoea is Passively Transferred to New-
Washington
Director
of TB
Corporation, 98119,
USA.
and Microbial 201
Elliott
Pathogens Avenue
Itesearch,
West,
Seattle,
571
Corporation,
Seattle, USA
In addition to the introductory demonstrations of genetic immunization, the past year has brought significant advances in vaccine development. Particularly encouraging are live recombinant vaccines, studies demonstrating the potential to elicit both systemic and mucosal immune responses, more studies demonstrating immune protection in animal disease models, and experimental
vaccines eliciting
immune
Current Opinion in Immunology
responses
1994,
in primates.
6:568-571
Introduction
live recombinant bacterial vaccine vehicles
Despite an ever increasing knowledge of immune mechanisms and protective antigens, the principal obstacles impeding the development of use&l and sail subunit vaccines have not changed significantly over the last 5 years. The enormous task of cost-effectively producing sufficient quantities of a potentially protective imrnunogen is one obstacle that often kills vaccine development efforts. The problems inherent in providing the immunogen in a safe format that still promotes the desired protective immune response are often insurmountable. Most vaccine delivery systems aim to minimize the impact of these major obstacles. Vaccine delivery systems can be roughly categorized as particulate vaccine carriers, live vaccine vehicles, and encoded vaccines. Particulate vaccine carriers (e.g. virus-like particles, liposomes and immune-stimulating complexes or ISCOMs) aim to enhance irmnunogenicity to such an extent that less of the immunogen is necessary to achieve the desired protective response. Such systems will not be covered in this review. Live vaccine vehicles, for example bacille calmette-guerin (BCG), Salmonella and pox viruses preclude the need to produce and purifjr protective immunogens, as these are expressed by the vehicle in the vaccinee upon introduction in viuo. The most recent strategy of genetic immunization employs encoded vaccines composed of the DNA or RNA of an immunogen. These encoded vaccines are directly introduced or transfected into tissues which then express the desired immunogen &om the encoded vaccine template, thereby obviating the need to design production and formulation processes for each immunogen. The past year has brought fLrther incremental advances in the understanding, and use, of live vaccine vehicles and the initial demonstrations of genetic immunization.
Among the bacteria being considered for live recombinant vaccine vehicles (LRWs), the most prominent are still attenuated Salmonella and mycobacterial BCG. While, previous studies have demonstrated protective responses to recombinant antigens expressed in attenuated Salmonella, the past year has brought the first reports of protective responses to recombinant antigens expressed in the recombinant BCG (rBCG) vaccine vehicle. A systematic comparison of antigen expression by rBCG within the cytoplasm, in the membrane as a chimeric lipoprotein, or as a secreted and cell wall associated protein, resulted in the construction of a promising vaccine against Lyme disease [ 1.1. Potent and lasting antibody responses were elicited to the OspA antigen of Ebwelia burgdotfeti, when it was expressed as a chimeric lipoprotein in the membrane of rBCG. These responses compared favorably with subunit vaccines based on OspA lipoprotein and are particularly noteworthy because it was anticipated that rBCG-based vaccines would be most suitable for eliciting cellular responses. It is likely that an rBCG-OspA vaccine will be the first one to be tested in humans. Also of note is an rBCG vaccine expressing a surf&e protein, PspA, of Streptococcus pneumoniae. Mice immunized with an rBCG-PspA vaccine showed protective humoral responses against virulent Pneumococcus and rBCG-PspA vaccines effectively primed with purified PspA-subunit vaccines (S Langermann, MS Hanson, JE Burlein, SR Palaszynski, LS McDaniel, DE Briles, CK Stover, unpublished data). A recent study with an rBCG vaccine expressing the gp63 antigen of Leishmaniu also indicates that protective cellular responses, which rBCG vaccines were originally intended for, can also be elicited to recombinant antigens expressed by rBCG [2].
Abbreviations BCG-bacille calmette-guerin; CTL-cytotoxic T lymphocyte; ISCOM-immune-stimulating MHC-major
568
complex; LRW-live recombinant vaccine vehicle; LT-B-heat-labile enterotoxin; histocompatibility complex; rBCC_recombinant BCG; W-simian immunodeficiency virus.
0 Current Biology Ltd ISSN 0952-7915
Recombinant vaccine deliverv svstems and encoded vaccines Stover
The first published studies with an rBCG vaccine in primates demonstrated specific MHC-restricted CD8+ cytotoxic T lymphocyte (CTL) responses to the Simian immunodeficiency virus (SIV)-mat gag epitope [3]. This study confirmed earlier findings [4] indicating a potential for rBCG to elicit class I restricted cellular responses even though BCG persists in the endosomal phagolysosome of macrophages and not in the cytoplasm where class I antigen presentation pathway is thought to operate. The first demonstration for the potential of rBCG to elicit antigen-specific mucosal responses was also reported in the past year (S Langermann, S Palaszynski, A Sadziene, S Koenig, CK Stover, unpublished data; [5]). A single intranasal immunization with rBCG-OspA or rBCG-PspA elicited substantial mucosal and systemic immune responses comparable to parental immunization with the same rBCG vaccines. This finding is particularly significant in comparison with Salmonella, which, as an enteric bacteria, is considered to be most promising for use as an oral vaccine for eliciting mucosal responses. Although BCG is still one of the most widely used vaccines in the world, one of the disadvantages of using rBCG vaccine is the controversy surrounding its efficacy against tuberculosis. Therefore, one of the most significant studies affecting potential rBCG vaccines is a retrospective statistical study that found the BCG tuberculosis vaccine to be significantly more effective than widely believed, particularly against serious forms of tuberculosis and mortality [6*]. The past year also brought incremental advances in the understanding and development of Salmonella as a live vaccine vehicle. To further enhance Sulmonella’s ability to elicit mucosal responses by oral immunization, expression vectors have been designed that permit easy &sion to heat-labile enterotoxin (LT-B) of Escheridh coli which take advantage of the mucosal imrnunostimulatory properties of LT-B [7]. These vectors allow fusions via a linker to the carboxy-terminal end of LT-B resulting in a pentameric LT-B antigen f&ion protein which should retain LT-B GM1 ganglioside-binding activity. Further study with LT-B antigen fusions in recombinant Salmonella vaccine vehicles is necessary to determine whether this combination of vaccine delivery strategies offers real advantages over each strategy alone. In a surprising study, Salmonellavaccines expressing higher levels of antigen horn an unstable plasmid vector, which was lost within 24 h of immunization, resulted in higher levels of antigen specific antibody (systemic and mucosal) than Salmonella expressing lower levels of antigen horn a much more stable (at least 21 days) single copy chromosomal vector [&I. This study suggests that the most important event in the development of an immune response against a foreign antigen delivered by Salmonella (and possibly other bacterial vector systems) may be the initial amount of antigen priming the immune response and not the persistence of the bacterial vector in tissues. This is somewhat surprising as considerable effort has been spent in developing stable vector systems for Salmonella and BCG to ensure longer term persistence of the bacterial vehicle.
live recombinant viral vaccine vehicles The most widely studied live vaccine vehicles are recombinant vaccinia viral vectors. Further progress was demonstrated in the potential for vaccinia to elicit substantial and relevant immune responses, and the potential for using recombinant vaccinia virus HIV-l antigens to prime for immunization with subunit vaccines or whole inactivated virus vaccines was reported in primate studies [9,10]. A study evaluating the effect of two different recombinant vaccinia virus immunizations on a single host revealed that priming with the first vaccinia virus can inhibit the titer and duration of an antibody response elicited by a second distinct recombinant vaccinia virus for nine months in mice [l 11. While vaccinia-based veterinary vaccines are being pursued, concerns about the safe use of recombinant vaccinia vectors have resulted in the engineering of the highly attenuated NYVAC vaccinia virus and the canary pox ALVAC virus for use as live vaccine vehicles [12,13]. Despite the drastic attenuation of NYVAC and the inability of the ALVAC virus to replicate in mammalian cells, evidence is mounting that these recombinant viruses can elicit strong and protective immune responses to extrinsic antigens expressed in these systems [14-161. Studies on the use of recombinant adenovirus as live viral vaccines proliferated in 1993. Recombinant adenovirus is thought to be particularly promising for generating mucosal immune responses. Promising systemic and mucosal immune responses to rotovirus VP7 and herpes simplex virus glycoprotein B antigens were demonstrated in two different studies using intranasal immunization with recombinant adenovirus [17,18]. In chimpanzees, recombinant human adenovirus expressing HIV-l env or gag-protease genes were used in multiple route inoculation regimens, which included boosting with subunit vaccines [19]. A variety of cellular and humoral responses were observed in this study including low-titered type-specific neutralizing antibodies and mucosal HIV-specific antibodies. New approaches to engineer hybrid live viruses and chimeric non-replicating pseudovirions have also been reported recently [20*,21]. It is likely that such approaches will proliferate in the near titure as the mechanisms of viral assembly for a variety of viruses are better understood.
Encoded vaccines and genetic immunization Perhaps the most noteworthy and controversial event in vaccine development in the past year was the first report of immune responses to genetic immunization using encoded vaccines. With this strategy of immunization, a genetic template (DNA or RNA) is directly injected into tissues, resulting in transfection of host cells and endogenous expression of the antigen encoded by the genetic template (encoded vaccine). This strategy eliminates the need to design specific processes for purification and formulation of different antigens and only requires the simpler and relatively standard method for purification of nucleic acid. A number of genetic
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immunization studies employing DNA encoding viral antigens or HIV-l or influenza viruses by intramuscular injection have demonstrated antigen-specific cellular and humoral immune responses .that were protective in mice and chickens or contained HIV-l viral neutralizing activity [22,23”,24]. Some of these studies indicate that DNA genetic immunization can be accomplished fairly efficiently by multiple routes of inoculation including mucosal routes [25*,26]. Another strategy of genetic immunization employed injection of liposome entrapped mRNA encoding influenza virus nucleoprotein (NP) [27]. In this study, MHC-restricted CTL responses to NP were elicited in mice of three different haplotypes. These CTL responses were very similar to those elicited by the live influenza virus in the same mouse strains. To elicit these responses, the mRNA encoding the NP antigen had to be encapsulated presumably because of m.RNA lability. While the preparation of mRNA and its encapsulation into liposomes adds two levels of complexity to the use of DNA for genetic immunization, the use of mRNA greatly reduces the potential risk of mutations resulting fi-om integration of viral DNA into the host genome. While these first genetic immunization studies have demonstrated the potential for encoded vaccines to elicit both cellular and humoral immune responses, f%rther characterization of such immune responses and comparisons with other vaccine delivery systems are necessary to evaluate the real potential for encoded vaccines.
Conclusions There are two basic problems confronting vaccine development: cost-effective production and purification of antigens f?om a pathogen or expression system in sufflcient quantities for vaccine studies; and the delivery of this antigen in a suitable and safe format (adjuvant or carrier) to promote protective immune responses. LRWs offer significant advantages over subunit vaccines because antigen immunogen purification is eliminated and the source of recombinant antigen replicates in the host, potentially providing a means of auto-boosting. An organism producing antigen is thought to be innately more immunogenic as the antigen is more likely to be recognized as foreign and processed by antigen-presenting cells. A variety of attenuated bacteria and viruses are currently under investigation as live vaccine vehicles. Despite the obvious advantages that LRWs potentially offer, they are generally not as palatable as subunit vaccines, especially in the USA, where no current live attenuated vaccines are used. Some of the reasons for this negative perception of LRWs may be more intuitive than factbased. It is a f&t that all humans carry commensal live replicating organisms, some of which are very beneficial. It also seems that many percieve the LRWs to be only a temporary solution or an inexpensive way to make vaccines for the developing world. While there are well founded concerns about the use of LRWs, particularly in immunosuppressed populations, we must also consider the problems associated with formulating safe immuno-
genic subunit vaccines and practical considerations, such as cost. Encoded vaccines seem to offer some of the advantages of a live vaccine without the organism vehicle. Concerns about eliciting anti-nucleic acid immune responses and the possibility that injecting large amounts of genetic material could cause mutations following insertional events in the host genome may be d&cult to overcome despite the available technology. As always, the benefits of new technologies must be weighed against risks which are both real and documented or perceived risks which may only be speculative. Some of the most significant progress to come in the next few years may be the availability of new technologies to deal with safety issues, however such advances usually lag behind, as in the case of pox virus delivery systems and the recent development of safer NYVAC and ALVAC viruses. The very latest developments in vaccine technology are reviewed in [28’,29*,30].Developments in gene therapy and approval of protocols for human trials may help pave the way for genetic immunization in humans. It is likely that we will see human clinical trials with some of the newer LRWs and encoded vaccine technologies in the next few years.
Acknowledgements I appreciate the helpful discussions on this topic with Dt TR Fuent of Genelabs Technologies, Dr S Langerman of MedImmune Incorporated, and Dr M Collett of PathoGenesis Corporation, and the assistance of S Colwell in the preparation of this manuscript.
References and recommended reading Papers of particular interest, published within the annual review, have been highlighted . of special interest .. of outstanding interest
period of
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1. .
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TF, Berkey CS, Wilson ME, Burdick E, Finebera HV, Mosteller F: Efficacy of BCC Vaccine in the Pre. vent%n”of kbefcubsis. j Am M&d Assoc 1994, 271:698-702. This study is a statistical meta-analysis of published clinical trials that BCC is 50% efficacious in preventing tuberculosis and 70-80% efficacious in preventing tuberculosis meningitis and death from tuberculosis. These findings are particularly significant because one of the major negatives aspects of the new rBCG vaccines is the controversy surrounding BCG’s efficacy against tuberculosis and its certain perturbation of surveillance procedures in the USA. In light of the TB resurgence, this study suggests that BCC immunization should be reconsidered.
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J, _Paoletti E: induction of Cytotoxic T LymRccombuunt Canarypox (ALVAC) and Attenuated kacchia (fiWAC) Viruses Expre&ngthe HIV-1 Envelope Clycoproteio. virology 1993, 195:845-850. P, Paoletti E: ProFelii leukemia Virus by Vaccination with a Canarypox Virus Recombinant, ALVAC-FL. / \/irol 1993,
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PathoGenesis
Rotaviru+lnduced Diirrhoea is Passively Transferred to New-
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