Nucleic acids: Vaccines of the future

Letters Under the Code, citation of authors’ names is always optional (Article 5 I a) because they do not form part of the scientific (zoological) names to which they are merely by convention - attached. Still, it would be worth proposing that a recommendation is incorporated Into the new Code to the effect that (it-respective of how many persons author the work as a whole) the number named against any new taxon should preferably not exceed four. This seems a reasonable maximum to me. (Among one’s concerns about super-multiple authorship is the needless recording burden placed on the biomedical databases. For my part, I begrudge those extra computer keystrokes.) Killick-Kendtick and Lainson’s worries stem partly from the insistence of some

journal edlton that describers’ names are given at least once in each publication for all cited genera/subgenera and species/ subspecles. This can be quite a burden where super-multiple authorship is involved and can make sequenced zoological names (interspersed with chains of authority names) difficult to read. The question IS: why do these editors require describer citation? It can hardly be so that the reader can easily track the orlginal description since a naked author’s name (without year date and blbliogtaphic reference) has pretty mlnimal source-finding value. To my mind, these editor; have got into a groove an empty ritual that has perhaps been perpetuated for a long time without its real point being questioned or brought to mind. Authority citation even in taxonomy

Nucleic

tends to be much overdone but It has a place in some kinds of speclallst taxonomic publication: it has little or no place elsewhere. Lastly, I clarify the status of KillickKendrick and Lainson’s transmogrification of poor old Uncle Tom’s surname from Cob(b)le into Cobbley. This was clearly unintentional (inadvertent would be the Code word) and so not an ‘emendatlon’ but an ‘Incorrect subsequent spelling’! Reference I Killick-Kendrick, R. and La~nson. R. Porasrtology Today IO, 468-469

(I 994)

Roger W. Crosskey Medical and Veterinary Divlslon Department of Entomology The Natural Histoy Museum London, UK SW7 5BD

Acids:

Vaccines of the Future G.J. Waine The recent successful immunization of experimental animals using nucleic sods has provided a revolutionary new approach in vaccinology. In this article, Gary Wa/ne and Don McManus examine the potential of nucleic acid vaccines for therr efictiveness not on/y against infectious and parasitic organisms exhibiting an intracellular phase during their life cycle, but also against parasitic helminths, whose life cycle stages are either predominant/y or completely extracellular. Nucleic acids look set to become the third generation of vaccines. First and second generation vaccines were primarily based on either of two technologies from which antigenic material was derived. These were: (I) live attenuated, or killed forms of whole organisms; and (2) defined native or recombinant protein components of the organism, obtained either by biochemlcal purification or by genetic engineering. Live attenuated vaccines, such as the polio and smallpox vaccines, stimulate protective cytotoxic T-cell (CTL) responses as well as T helper (Th)-cell and humor-a (antibody) immunity. However, a major concern with live vaccines is the intrinsic risk, however remote, of reversion to a pathogenic forrr’,2. Killed vaccines do not carty this risk, but, while such vaccines can generate Th-cell and humoral immune responses against a pathogen, they are usually unable to Porositology Toduy, vol. / I, no. 3, I995

and D.P. McManus

generate specific CTL responses3. Similarly, vaccination with defined protein components, such as tetanus or diphtheria toxoid, or the recombinantly derived hepatitis B surface antigen, induces Th-cell and humoral immune responses but generally not CTL responses. Nucleic acid vaccines* are able to induce all these responses, including specific CTL as well as Th-cell and humor-a responses4 6, without the intrinsic risks associated with live vaccines. The technology is relatively simple. A DNA (or RNA) construct encoding the gene of interest is delivered directly to cells of the organism to be immunized, where it is taken up and expressed by the host cells. The endogenously expressed immunogen subsequently induces a protective immune response in the host. Methods of Administration Numerous approaches for the delivery of foreign genes into mammalian somatic tissues or organs have been reported with varying degrees of success. These include in vivo infection using recombinant retroviral vecton7,8, encapsulation of DNA in liposomes9,‘0, delivet-y of DNA complexed with specific

*

Terminology recommended at a Meeting on Nucleic Acid Vaccines held on 17-l 8 May, 1994, World Health Organization Headquarters Geneva. Switzerland. Q I995 Elsewr

Science Ltd 0 I69

4758/95/$09

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protein carriers I, and delivery of plasmid DNA alone3 68~214. A number of these procedures have been shown to elicit immune responses. The first report describing the successful use of plasmid DNA as a vaccine was published by Ulmer and colleagues4. A plasmid DNA construct, encoding the nucleoprotein (NP) of influenza A virus, was injected directly into the quadriceps muscles of mice. This vaccine successfully protected 90% of the mice against a subsequent lethal challenge with influenza A virus, while only 20% of the controls survived. Both NPspecific CTLs and high-titre NP-specific IgG antibodies were detected in these experiments, although the protective immune response was demonstrated not to be mediated by the NP-specific antibodies, and was thus probably due to NP-specific cellular immunity. Generation of high-titre IgG antibodies is thought to require CD4+ Th cellsl5. Indeed, spleen-cell proliferative responses, which typically reflect activation of Th cells, have been observed following vaccination with plasmid DNA6 Thus, injection of nucleic acids can generate CTL as well as Th-cell and specific antibody responses, although, depending on the pathogen, not all of these responses may be required for protection. Other reports have also shown the efficacy of nucleic acid injection in inducing protective immune responses. 113

In one report, injection of an H7 haemagglutinin-expressing DNA was shown to protect chickens against lethal challenge with H7N7 influenza virusI6. Protective responses have also been obtained in cattle injected with plasmids encoding bovine herpesvirus I (BHV-I) glycoproteins3. The vaccinated calves developed significant antibody titres to the glycoprotein glV and shed less virus than did the control calf after challenge. Direct injection of DNA has also been shown to induce neutralizing antibodies against human immunodeficiency virus type I (HIV-I) in both mice and nonhuman primates6,13. Recently, the effectiveness of another technology, termed particle bombardment-mediated gene transfer, to introduce biologically active foreign genes into mammalian tissues has been demonstrated. In this technique, DNAcoated gold particles are accelerated to high velocity, facilitating efficient penetration of target organs and tissues, in viv01~m’~.Delivery of DNA to the epidermis of live mice by particle bombardment was shown not only to induce a specific humor-al immune response, but to actually protect the mice from subsequent lethal challenge with virusI9. Another, and potentially even more practical route of vaccination, successfully shown using nucleic acid vaccines, is immunization via mucosal tissues, This approach requires no specially trained personnel or equipment (such as a syringe or gene gun) for delivery. Fynan and colleagues I9 have shown that the same nucleic acid vaccine successfully used in their particle bombardment experiment was also able to protect mice (76% survival) against lethal challenge when it was administered intranasally as DNA drops. The ability to deliver nucleic acid vaccines in the form of nose drops or even a nasal spray clearly has important implications for future development and delivery of such vaccines. Advantages and Safety Issues Nucleic acid vaccines have several major advantages over other types of available vaccines. First, as previously discussed, nucleic acid vaccines can generate CTL responses without the use of replicating vector or live organisms. A second important advantage is that in SI~Uexpression of genes by the host cells should lead to synthesis of proteins that more closely resemble the native molecule, compared to those likely to be generated in prokaryotic expression systems (unless of course, the infectious agent is a prokatyote). Such 114

immunogens should, therefore, contain conformationally relevant epitopes and may be able to induce a more-effective immune response. Expression of protein following administration of plasmid has been shown to penis-t, with two reports having measured continued expression for at least I 8 months after administration 1a,20. Specific antibody levels have been shown to remain elevated in some animals for at least a year, with no decrease in titre5. Such observations raise the possibility of stimulating long-term immunity from a single nucleic acid vaccine dose, although it should be emphasized that successful, sustained protection has yet to be proven. It seems likely that the production costs for nucleic acid vaccines would be significantly less than those for, say, a recombinantly derived polypeptide vaccine, as the expense and problems associated with large-scale production of protein antigens (cell culture, fermentation, purification) are avoided. A further potential advantage of plasmid DNA vaccines is that the DNA can be processed to a dried pellet for simplified storage and transport. A dried DNA pellet would be relatively stable at ambient temperatures, potentially eliminating the requirement for the ‘coldchain’, and hence simplifying access to geographically remote areas. Ultimately, the DNA could be reconstituted simply by adding water immediately prior to administration. This would clearly be an advantage economically and in terms of public health. As a counterpoint to these advantages, the use of nucleic acid vaccines for innoculation raises a number of theoretical safety issues that need careful consideration. One concern is the unknown consequences of long-term persistence of plasmid DNA and foreign gene expression in the host. Such consequences include the possibility of inducing tolerance, autoimmunity, anaphylaxis (or hyperimmunity), as well as the possibility of inducing anti-DNA antibodies. Another theoretical concern is the possibility of vector DNA integrating into the host genome, resulting in an untoward transformation event. This could be caused by the insertlon of an oncogene, insertional activation of a host proto-oncogene, or insertional deactivation of a host suppressor gene. In one study, junctions between chromosomal and plasmid DNA that would indicate chromosomal integration were searched for by electroporating E. CO/I with injected, muscle DNA that was cut and re-ligated2Q. No

such integration events were seen in over I800 plasmids screened. In that study, the bacterial methylation pattern of plasmid DNA injected into muscles did not change, even after being maintained in the muscle for I9 months, indicating that plasmid replication did not occur. Regarding anti-DNA antibodies, a study in primates was unable to detect anti-nuclear or anti-DNA antibodies, even after repetitive DNA administrations21. However, to validate the safety of nucleic acid vaccines, further long-term investigations are needed. mRNA One possibility for circumventing these particular safety questions would be to use mRNA, instead of DNA, as it is transient in nature, does not persist, will not integrate into chromosomal DNA and will not cause insertional mutagenesis. Injection of mRNA direaly into mouse skeletal muscle has been shown to result in transient expression of reporter genes in vivol2. Subcutaneous and intravenous (but not intraperitoneal) administration of influenza-A NP mRNA encapsulated in liposomes has been shown to elicit NP-specific CTL responses22, indicating that, like DNA, mRNA is able to induce specific immune responses. Injection of mRNA alone, however, was not successful, although intro-muscular injection was not tested. Recently, particle bombardmentmediated gene-transfer technology was shown to be an efficient and reliable means for RNA delivery into various mammalian somatic tissues23. Transgene expression of RNA transcripts of three reporter genes [firefly luciferase (luc), human growth hormone (hGH), and human alpha- I antitrypsin (hAAT)] were detected in mouse epidermal or rat liver tissues bombarded in viva. The production of high-titre antibodies against hAAT, in VIVO,was detected in the mice following in situ bombardment of epidermal skin tissue with hAAT mRNA. It should be emphasized that mRNA does not provide all the advantages of DNA (see above). Transient expression is less likely to induce long-term expression and, therefore, one dose may not be sufficient to generate long-term immunity. Furthermore, mRNA is less stable than DNA, and the costs associated with its production, storage and transportation are likely to be higher, Cellular and Humoral

Responses

The various immune responses generated by nucleic acid vaccines are Porasrtoiogy

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Focus Fig. I. A proposed model for induction of protective immunity by nucleic acid vaccines. Successful methods for delivering nucleic acids to host tissues in vivo include direct injection (usually into muscles), particle bombardment using a gene gun (into epidermal tissue) and intmnasal administration of DNA drops. By mechanisms not fully understood, nucleic acids (plasmid DNA or mRNA) are taken up into the host cell. Plasmid DNA must be taken up into the nucleus for expression to occur, while mRNA needs only to reach the cytoplasm. The endogenously synthesized protein could then interact with the host immune system via MHC class I to generate specific CTLs, or, following release into the extracellular environment, with antigen presenting cells (APC) and Th cells, leading to specific antibody production by B cells.

Host cell Delivery

Injection

WH

Gene gun

t

Nose-drops )

likely to arise by different mechanisms. A proposed model is depicted in Fig. I. CD8+-restricted T cells (CTLs), whose main function is to recognize and kill virally infected cells, recognize antigens as peptides associated with the host cells MHC class I antigens. Peptides that associate with MHC class I molecules are generally endogenously derived by synthesis in the cytoplasm. From here they are transported into the lumen of the endoplasmic reticulum where they are available for co-assembly with the MHC class I heavy chain and p2 microglobulin24. The p2 assembled complex is then translocated to the cell plasma membrane, where it can become the target for immune surveillance by CD8+restricted 0-s. This is the normal mechanism for identification and destruction of virally infected cells, and would seem to be the probable mechanism by which specific CTLs are generated when nucleic acids are taken up and endogenously expressed by the host cells. But how would Th cells (CD4+restricted T cells) and antibody (B-cell) responses be generated? One possible mechanism is that small amounts of the endogenously synthesized peptide could be shed from the transfected cell continuously, or released following cell death. The extracellular protein could then be endocytosed into peripheral endosomal compartments by ‘professional’ antigen-presenting cells (APCs), and subsequently processed and asParasitology Today, vol. I I, no. 3, I995

of plasmid

DNA to host cells

Protein released

sembled with the alpha and beta chains of MHC class II molecules, to form a mature MHC class II-antigen complex. After translocation to the cell surface, the assembled complex would then become a target for immune surveillance by CD4+-restricted Th cells, which, in turn, provide help for antibody production by B cells. Nucleic Acid Vaccines against Parasitic Infections Nucleic acid vaccines have been examined predominantly in relation to viral infections, which replicate inttacellulariy. The ability to generate specific CTLs against such infected cells is a clear benefit of nucleic acid vaccines. However, nucleic acid vaccines may also be effective against parasitic organisms, whose life cycle stages are either predominantly or completely extracellular. Protective immunity to these infections is likely to involve specific antibodies and Th cells, rather than CTLs. Our laboratory is investigating the use of nucleic acid vaccine technology particularly in relation to schistosomiasis. The basis for our approach is that, although CD8+ CTLs appear not to be involved in protection against schistosomiasis, at least in micezs, there is considerable evidence that antibodies help mediate protection against schistosomiasis via antibody-dependent cellular cytotoxicity (ADCC) reactions. For example, recent studies in both mice2@* and baboons29

suggest that protective immunity is antibody dependent. In human populations, immunoepidemiological studies have shown a close correlation between the acquisition of protective immunity to schistosomiasis and the level of specific IgE and IgA antibodies against schistosome adult worm antigens3&x3. The anti-schistosome response is likely to involve specific antibodies in association with effector cells such as macrophages, platelets and eosinophils. A high-affinity IgE receptor (FceRI), expressed on eosinophils from hypereosinophilic Schistosoma monsoni patients, is involved in eosinophil degranulation, and participates in eosinophil-mediated cytotoxicity against Schisrosoma manson?. It is likely that a nucleic acid vaccine, which can generate appropriate Th-cell and antibody responses, would induce protective antibody-dependent cell-mediated immunity against schistosomiasis. Our laboratory is endeavouring to develop such a vaccine against 8. japonicum using a number of S.japonicum cDNAs we have recently cloned35-39, many of which encode homologues of 5. mansoni antigens known to be potential vaccine candidates for schistosomiasis mansoni. In addition to schistosomiasis, nucleic acid vaccines may also be effective against a range of other parasites. Indeed, promising results using nucleic acids to vaccinate experimental animals against leishmaniasis and malaria have recently been reported40,41. A further 115

Focus significant benefit of this technology is that it may assist in the identification of protective antigens for parasitic and other diseases for which protective antigens have not yet been identified, by greatly simplifying the process required to test them in experimental animals. Acknowledgements We would like to thank Bemd Kalinna for kind assistance with the production of Fig. I. Our studies into the development of antischistosomiasis vaccines including nucleic acid vaccines receive financial support from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases, and the National Health and Medical Research Council of Australia. References Ogra. P.L. et al. (199 I) J. infect 0s. 164, 191-194 Weeks-Levy, C. et al. (199 I) V~roiogy 185, 934-937 Cox. G.J. et al. (I 993)]. Viral. 67, 5664-5667 Ulmer, J.B. et al. (1993) Science 259, 1745-1749 Rhodes, G.H. et 01. (1993) Vacones 93, 137-140 Wang, B. et al. (I 993) Proc. Not\ Acad. Ser. USA 90.4 156-4 I60 Dubensky, T.W. et al. (I 984) Pmt. Nat/ Acad. SC;.USA 8 I, 7529 -7533 Hunt, L.A. et al. (1988) 1, V~roi. 62, 3014~3019 Kaneda, Y. et al. (I 989) Science 243, 375-378

IO Zhu, N. et ai. (I 993) Scrence 26 I, 209-2 I I Wu, C. et al. (I 989) J. 8101. Chem. 264, 16985-16987 J.A. et a/. (I 990) Scrence 247, I2 Wolff, 1465-1468 I3 Wang, B. et a/. (1993) DNA Cell. Biol. 12, 799 -805 I4 Geissler, E.K. et ai. (1994) 1. immunol. 152, 413-421 15 Vleira, P. and Rajewsky, K. (1990) lnt lmmunol. 2.487-494 16 Robinson, H. et a/. (I 993) Vaccrne I I, 957-960 I7 Yang, N-S. et al. ( 1990) Proc. Nat\ Acad. Sci. USA 87,9568-9572 18 Cheng, L. et al. (I 993) Proc. Natl Acad. Sci. USA 90,4455-4459 I9 Fynan, E.F. et al. (1993) Proc. Nati Acad. Sci. USA 90, I I 478- i 1482 20 Wolff, J.A. et al. (1992) Hum. Mol. Gen. I, 363-369 21 Jiao, S.M. et ai. (1992) Hum. Gene Ther. 3, 21-33 22 Martinon, F. et al. (I 993) Eur. 1. immunol. 23, 1719-1722 23 Yang, N. et ai. (I 994)/. Cell. Blochem. (Suppl.) I8A, 230 24 Getmain, R.N. and Margulies, D.H. (1993) Annu. Rev. immunol. I I, 403-450 25 Vignali, D.A. et al. (1989) immunology 67, 266-472 26 Moloney, N.A. and Webb, G. (I 990) Parasrtology IOO,235-239 27 Delgado, V. and McClaren, D.J. (I 990) Parasite lmmunol. 12. I S-32 28 Xu. C-B. et al. (1993) 1, lmmunoi. 150, 940-949 29 Soisson, L.A. et al. (1993) 1. Immunol. I5 I, 4782-4789

30 Hagan, P. et al. (I 99 I) Nature 349, 243-245 3 I Rihet, P. et al. (I 99 I) Eur. J. lmmunol. 21, 2679-2688 32 Dunne, D.W. et ai. (I 992) Eur.]. immunol. 22, 1483-1494 33 Grzych, J-M. et al.(1993) 1. Immunol.150. 527-535 34 Gounni. A.S. et al. (1994) Nature 367, 183-186 35 Yang, W. et ai. (1992) int J. Parositol. 22, I l87-! I91 36 Waine, G.J. et al. (1993) Infect. Immun. 61, 47 16-4723 37 Waine, G.J. et ai. (1993) Blochem. Blophys. Res. Commun. 195, I21 l-1217 38 Waine, G.J. et al. ( 1994) Gene 142, 259-263 39 Becker, MM et ai. (1994) Gene 148, 321-325 40 Waine, G.J. (1994) Parasrtology Today IO, 453-454 4 I Sedegah, M. ( 1994) Proc. Nat/ Acad. Ser. USA 9 I, 9866-9870

Note added in proof The entire issue of Vaccine I2 (I 6), 1994, is devoted to a WHO meeting on nucleic acid vaccines, and includes reports on the development of nucleic acid vaccines against leishmaniasis and malaria. Gary Waine and Don McManus are with the Tropical Health Programme, Molecular Parasitology Unit Queensland Institute of Medical Research, 300 Henton Road, Brisbane, Queensland 4029, Australia. Tel: +617 362 0400, Fax: +617 362 01 I I, e-mail: [email protected]

The Microfilarial Sheath and its Proteins H. Zahner, The

microflarbe

filarioe

of

ore enclosed

several

genera

by a flexible,

of bag-

like structure, the ‘microfilariol sheath: which is synthesized in a co-ordinated way by both the embryo and the uterine epithelium. In this article, Horst Zohner, Gerd Hobom and Stephan Stirm consider its structureand composition, and its role in the survivalstrategyofthe parasites by evading the host’s immune reaction.

Filatiae are viviparous nematodes which release their firs-stage larvae, the microfilariae, into the host’s tissue, where they will persist for weeks or months. In Wuchereria, Brugia, litomosoides and some other genera, the microfilariae are enclosed by a microfilarial sheath (Fig. I). In these cases, it is not the cuticle but the sheath surface that displays the host-parasite interface, as the sheath is impermeable to larger proteins such as antibodies. Consequently, its surface antigens appear to be involved in immune elimination, and are generally not II6

G. Hobom

and S. Stirrn

recognized in microfilaraemic hosts’. Surface exposure of host-related antigenie structures of microfilarial proteins or protein modifications appears to be the most likely explanation for such a kind of mimicry. Attematively, the sheath may contain host serum proteins adsorbed to its surface (albumin and/or immunoglobulins in species-dependent compositions2,3), but it is uncertain whether these are of any major importance in parasite-host interactions. Structure

and Composition

The microfilarial sheaths of the various genera and species have very similar structures. There is clearly a common formation and morphology. All of them consist of two major structural components4-6; an internal homogeneous layer, which may be regarded as a modified remnant of the primary egg shell, and an external particulate layer, which is accumulated from cellular ex0

1995.Elsev~er Scence Ltd 0169%4758/951$09,50

cretions of the distal part of the uterus at the surface of the homogeneous layer 4.5. The structura composition of the microfilarial sheaths remained largely unknown for a long time due to the unavailability of sufficient amounts of material; this changed when techniques were developed7,8 that allowed the isolation of pure, intact sheaths. In the case of Litomosoides carinii, approximately 55% of the sheath dry mass consists of proteins with glutamine and proline as the major amino acids8 (Table I). In Brugia spp, weight-related determinations of sheath components were not possible because only much smaller amounts of material were available, but relative ratios of amino acid compositions turned out to be almost identical with L. coriniP. Total carbohydrates account for approximately 8% of weight (Table I), but neither the mature sheaths of 1. carinii nor of Brugio contain appreciable amounts of glucosamine, ie. no chitin is Parasitology

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