- Email: [email protected]
Vaccines for parasitic and bacterial diseases
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Vaccines for parasitic and bacterial diseases Steven G Reedy and Antonio Campos-Netoz The first decade of the millennium should mark the beginning of a new era in vaccine development, reaping the rewards of advances in genome characterization, antigen identification, understanding the molecular bases of protective immune responses, and adjuvant design and development. Advances in all of these areas have culminated in vaccine candidates entering clinical testing. These include vaccines against two of humankind’s oldest and deadliest diseases, tuberculosis and malaria. Several vaccine candidates for each of these diseases will be tested in humans during the next few years. A candidate vaccine for leishmaniasis, an infection that has taught us much about T-cell regulation of protection and disease in animal models, has been developed and is now in the clinic. There are indications both in animal models and in patients that vaccines may be used not only to protect but also to treat leishmania infections. Addresses Infectious Disease Research Institute, 1124 Columbia Street, Suite 600, Seattle, WA 98104, USA y Corixa Corporation, 1124 Columbia Street, Suite 200, Seattle, WA 98104, USA; and, University of Washington, Department of Pathobiology, Box 357238, Seattle, WA 98195, USA e-mail: [email protected] z e-mail: [email protected]
Current Opinion in Immunology 2003, 15:456–460 This review comes from a themed issue on Host–pathogen interactions Edited by Robert L Modlin and Peter Doherty 0952-7915/$ – see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0952-7915(03)00069-4
Abbreviations BCG bacillus Calmette-Gue´rin IFN interferon MPL monophosphoryl lipid A MPL-SE MPL stable emulsion
Introduction The pace of vaccine development for complex organisms has been accelerated in the past decade because of advances in antigen discovery including genome sequencing, increased understanding of immune mechanisms of protection and, perhaps most importantly, the development of safe and effective vaccine adjuvants and delivery platforms. Only a few years ago it was thought that protective T-cell responses to intracellular pathogens could be achieved only by immunization with live vaccines or by using adjuvants deemed unacceptable for humans because Current Opinion in Immunology 2003, 15:456–460
of reactogenicity. Over the past several years it has become evident that a new generation of vaccines is feasible. Most of these vaccines will consist of defined antigens, and many will involve new adjuvants or delivery technologies capable of stimulating a broad range of immune responses. In this brief review, we will focus on a few infectious agents for which significant advances in vaccine development have been made very recently.
Malaria Significant progress has been made on moving promising candidates into development for clinical trials. One candidate that has moved into the clinic is the 19 kDa merozoite surface antigen (MSP-1) of Plasmodium falciparum. This protein was tested as an alum-adsorbed vaccine in a Phase I trial of normal volunteers, in whom induction of a range of cytokines was detected [1]. The importance of MSP-1 was further documented in a study demonstrating that antibodies to this surface protein were important in blocking parasite invasion of erythrocytes in individuals immune to malaria [2]. One of the more interesting advances in malaria vaccine development is the use of parasite antigens coupled to particulate viral proteins as a way to induce potent immune responses. The RTS,S vaccine developed by GlaxoSmithKline Biologicals (Rixensart, Belgium) and collaborators includes the P. falciparum circumsporozoite (CS) protein expressed in a hepatitis B surface antigen particle to increase immunogenicity. The vaccine is delivered in ASO2, an adjuvant that contains monophosphoryl lipid A (MPL1; Corixa Corporation, Hamilton, MT, USA) and QS-21. The vaccine was previously shown to induce partial protection in human challenge experiments and more recently has been tested in African field trials where significant, although not long-lasting, protection was reported [3,4]. Another approach to induce potent immune responses to the CS protein was used by Birkett et al. [5], who expressed B- and T-cell epitopes of this protein in a hepatitis B core protein. This construct, termed ICC-1132, was highly immunogenic when formulated with alum or in Montanide ISA 720. This approach may be applicable for antigens for which epitopes of limited size can be inserted into the viral core protein. The ICC-1132 vaccine is scheduled for clinical trials soon.
Leishmaniasis The intracellular parasites belonging to the Leishmania complex have been used for decades in vaccine models for diseases requiring potent T-cell immunity. A few antigens that induce high degrees of protection have been identified, but until recently no practical adjuvants www.current-opinion.com
Vaccines for parasites and bacteria Reed and Campos-Neto
or delivery systems were available to move vaccine candidates into the clinic. A mixture of three antigens (thiol-specific antioxidant [TSA], Leishmania major stressinducible protein-1 [LmSTI-1] and Leishmania elongation initiation factor [LeIF]) has been found to induce protection in mouse and monkey models when delivered in IL-12 or as DNA [6,7]. Other adjuvants have been evaluated in the BALB/c mouse model, including CpG and MPL1 stable emulsion (MPL-SE). The three vaccine antigens have been produced as a single fusion protein, Leish-111f, which conferred excellent protection as DNA or protein [8,9]. A candidate vaccine consisting of Leish-111f formulated in MPL-SE entered Phase I clinical testing in normal volunteers in January 2003. The three antigens that make up Leish-111f have been used to develop an immunotherapeutic strategy to treat patients with refractory mucosal leishmaniasis [10]. This report follows previous studies in which crude parasite preparations, either alone or with the live attenuated strain of Mycobacterium bovis — bacillus Calmette-Gue´ rin (BCG), were administered to patients with mucosal leishmaniasis. The earlier studies with crude vaccine preparations, and later with defined antigens, have demonstrated that vaccines can be used to alter the course of an ongoing disease characterized not by an anergic state, but rather by hyperreactivity to parasite antigens. In a recent study, we examined T-cell responses to parasite antigens pre- and post-immunization with the recombinant proteins that make up Leish-111f. Preliminary data from our laboratory indicates that, although patient peripheral blood mononuclear cells (PBMCs) proliferated strongly in response to parasite lysate before immunization, the recombinant antigens elicited much weaker proliferative responses in most cases. Not surprisingly, IFN-g production in response to the recombinant antigens increased following immunization. The most significant finding, however, was a marked decrease in IL-5 production in response to parasite lysate following immunization with the recombinant proteins. Although this response was high before immunization, it decreased to background levels after the third vaccine dose. The decrease in IL-5 production correlated with improvement in, or resolution of, clinical signs and symptoms. These observations support the concept that a T-cell vaccine can be used to alter an ongoing immune response and consequently to treat disease. Understanding the properties of a vaccine that are responsible for this will help in the design of effective immunotherapeutics. One plausible mechanism is the induction of IL-12 by LeIF, one of the components of the vaccine. LeIF stimulates potent innate immune responses, and should perhaps be considered as an important component of an effective vaccine or immunotherapeutic [11]. Another exciting and controversial approach is vaccinating against vector-borne diseases, as reported by Sacks, www.current-opinion.com
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Ribeiro and colleagues [12]. To achieve protection against leishmaniasis, this group immunized mice with naked DNA encoding a protein from the salivary gland from the sandfly vector instead of with proteins from the parasites [12]. Solid protection accompanied by high IFN-g production was observed after natural challenge of the mice using infected sandflies. Protection was presumably conferred in part by an inflammatory process mediated by immune recognition of the fly components. The implications of this approach for vaccine development have been discussed elsewhere [13].
Mycobacterium tuberculosis Approximately one-third of the world’s population is infected with M. tuberculosis and it is estimated that this disease causes three million deaths each year. Despite the effective treatment of tuberculosis with a combination of three antibiotics the treatment is long, resulting in inadequate compliancy and leading to the development of several multidrug-resistant strains of M. tuberculosis. Compounded with the emergence of the M. tuberculosis-HIV co-infection epidemic in both developing and industrialized countries, this underscores the need for an effective vaccine against the disease. The current and only available vaccine, BCG, although efficacious in preventing the less common yet severe forms of the disease in young children (meningitis and systemic disease) has no effect against adult pulmonary tuberculosis, the most common form of the disease. A serious complicating factor is our lack of a clear understanding of the mechanism of protection in tuberculosis. It is clear, however, that the CD4þ T-cell response plays an important role in immunity. CD8þ T cells may also participate in resistance but in a more restricted manner, perhaps keeping the latent phase of the infectious process in check. The strong T-cell response to CD1-associated non-protein antigens of M. tuberculosis observed in many sensitized individuals, though intriguing and unique, has not proven to be involved in the immune defense mechanisms against tuberculosis [14]. More recently, another unique system has been described for T-cell recognition of M. tuberculosis antigens [15]. CD8þ T cells from people with latent tuberculosis infection recognize pathogen antigens in complex with HLA-E, as well as in complex with the more conventional HLA-A, -B, or -C MHC class I molecules. The implication of these findings for resistance to tuberculosis has not yet been studied. Over the past decade several investigators have concentrated major efforts in the development of a better vaccine than BCG. A number of different approaches have been pursued. Most have focused on the development of a recombinant subunit vaccine (protein or plasmid DNA), while others have concentrated either on the improvement of the BCG vaccination or in the development of viable attenuated M. tuberculosis. The molecular Current Opinion in Immunology 2003, 15:456–460
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manipulation of BCG, by insertion of either mammalian genes encoding cytokines (aiming to enhance or modulate the immune response induced by the bacteria) or M. tuberculosis genes encoding proteins of the tubercle bacilli, resulted in mycobacterial variants that at best, marginally augmented the protective efficacy of the original BCG strain. However, some success has recently been reported with recombinant BCG strains (see also Update). In addition, genetic manipulation of virulent M. tuberculosis has generated interesting auxotrophic mutants with low virulence and protective efficacy similar to that induced by BCG in mice challenged with the virulent bacilli [16–19]. Despite the fact that the protection induced by these auxotrophic mutants has not surpassed BCG, this approach resulted in a highly protective vaccine. Viable attenuated microorganisms are the components of highly efficient vaccines against several human infectious diseases, including smallpox, polio, measles, mumps and leishmaniasis. Attenuation of virulence of the microorganisms present in these vaccines resulted either from an accident of nature (e.g. smallpox) or from empirical manipulations of the microbe cultures. The idea and feasibility of using rational and modern molecular genetic approaches to engineer a non-virulent M. tuberculosis strain that preserves most of the antigenic repertoire of the virulent strain is highly attractive and has the potential to generate a viable anti-tuberculosis vaccine that is superior to BCG. Much progress has also been made in the development of an anti-tuberculosis recombinant subunit vaccine. In particular, bacterial plasmid DNAs containing M. tuberculosis genes have been extensively tested over the past ten years. However, the initial euphoria associated with this method of antigen delivery has been hampered by an inability to reproduce the exciting original results beyond the murine model of tuberculosis. A plethora of publications describing the protection of mice, induced by many different M. tuberculosis DNAs, could not be reproduced in either the guinea pig or monkey models. Despite several attempts at optimizing codon usage and alternating priming/boosting with either BCG or adenovirus carrying the M. tuberculosis genes, there was little evidence of improvement. More encouraging, however, are the results obtained with conventional protein immunization formulated with newer adjuvants capable of both amplifying and modulating the immune response to the T helper 1 (Th1) phenotype. Examples are the MPL1 and a series of adjuvants containing MPL1 in association with CpG oligonucleotides (AS02 adjuvants from GlaxoSmithKline). Importantly, these adjuvants are safe for human use [3]. Somewhat unexpectedly, these approaches promote the emergence of CD8þ-specific T-cell responses to the protein antigen [20,21]. As mentioned above, both Th1 CD4þ T cells and CD8þ T cells are important Current Opinion in Immunology 2003, 15:456–460
mediators of immunity against tuberculosis. Also encouraging are the results obtained with recombinant polyproteins of M. tuberculosis. Such approaches (i.e. fusing the genes encoding proteins that induce partial protection) result in single larger molecules that preserve both the immunogenicity and protective efficacy of each individual component of the polyprotein [22]. Excellent shortand long-term protection has been obtained using such molecules formulated with the adjuvant AS02 (GlaxoSmithKline) in mice, guinea pig and monkey models of tuberculosis [23]. In conclusion, the development of both interesting auxotrophic mutants of M. tuberculosis (viable attenuated vaccine) and recombinant polyproteins of the tubercle bacilli associated with unique adjuvants (subunit vaccine) should ultimately result in a more efficacious vaccine than the current BCG.
Streptococcus pneumoniae and Neisseria meningitidis Streptococcus pneumoniae is the leading etiological agent of bacterial pneumonia, meningitis and otitis media in both adults and children. Approximately one million children under the age of five die every year from the pneumonia caused by S. pneumoniae. Neisseria meningitidis causes endemic and epidemic bacterial meningitis. The serious and frequent diseases caused by these organisms occur in both developed and developing nations. In contrast to M. tuberculosis, S. pneumoniae and N. meningitidis are extracellular infectious agents, and immunity to them is primarily mediated by specific antibodies to the bacterial surface antigens. Both organisms produce extensive polysaccharide capsules, which are the antigenic target of existing vaccines against these bacteria. However, one serious complicating factor with these vaccines is the immense antigenic heterogeneity of the capsular polysaccharide of both organisms. For example, there are at least 23 common serotypes of S. pneumoniae that cause disease in humans. Similarly, N. meningitidis can be segregated into at least 13 serogroups. The available vaccines for use in the United States against S. pneumoniae contain a mixture of 25mg of polysaccharide of each of the 23 prevalent serotypes. Currently available vaccines against N. meningitidis include a quadrivalent product containing the polysaccharides of groups A, C, Y and W-135. No vaccine is available for group B because, for unknown reasons, this polysaccharide is not immunogenic in humans. Another serious complicating factor with these vaccines is the polysaccharide nature of their antigens. Because the immune response to such antigens is T-cell independent, immunological memory is not elicited and there is thus no boosting effect with repeated immunizations. The alternative approach of conjugating a microbial protein carrier to the polysaccharide has slightly improved the efficacy of the anti-polysaccharide C group of N. meningitidis. Thus far, no convincing data exists to show improvement in either antibody titers or the induction of memory to S. pneumoniae polysaccharide. www.current-opinion.com
Vaccines for parasites and bacteria Reed and Campos-Neto
Although the concept that ideal vaccines against microbes for which immunity is antibody mediated should include surface proteins that are conserved across all the serotypes/serogroups is not new, the identification and characterization of such molecules has been difficult. Notwithstanding, one such molecule has recently been described [24]. More importantly, another recent report describes an interesting general approach for the identification, screening and cloning of the genes encoding bacterial cell-surface peptides [25]. A Staphylococcus aureus genomic DNA library was initially generated using short DNA fragments ranging from 150 to 300bps. To facilitate cell-surface expression, the DNA fragments were digested with restriction enzymes and fused with the genes encoding one of the two outer membrane proteins LamB and FhuA. The use of high-titer human sera from patients suffering from moderate to severe S. aureus infections, in combination with magnetic cell sorting and an opsonophagocytosis assay to screen the library, resulted in the cloning and sequencing of several genes. Alignment of these sequences with the S. aureus genome revealed that several of the corresponding fulllength genes encoded proteins that were either located on the surface of the bacterium or secreted. An antibody specific for one such peptide revealed its presence on the cell surface of S. aureus and, more importantly, facilitated the phagocytosis of these organisms by human polymorphonuclear cells. This approach should therefore be of great value for the identification of protein vaccine candidates for pathogens, where immunity is antibody mediated and the complete genome sequence has been determined, as in the case of S. pneumonia and N. meningitidis [26–28].
begun, so that we can now begin to learn how to build successful vaccines for these challenging diseases.
Update Recent work has shown that recombinant BCG expressing ESAT-6 or the 30 kDa secretory protein confers enhanced protection against challenge with virulent M. tuberculosis [29–31].
Acknowledgements This work supported in part by National Institutes of Health grants AI-25038, AI-43528, AI-44373, and AI-49505.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
Lee EA, Palmer DR, Flanagan KL, Reece WH, Odhiambo K, Marsh K, Pinder M, Gravenor MB, Keitel WA, Kester KE et al.: Induction of T helper type 1 and 2 responses to 19-kilodalton merozoite surface protein 1 in vaccinated healthy volunteers and adults naturally exposed to malaria. Infect Immun 2002, 70:1417-1421.
2.
O’Donnell RA, Koning-Ward TF, Burt RA, Bockarie M, Reeder JC, Cowman AF, Crabb BS: Antibodies against merozoite surface protein (MSP)-1(19) are a major component of the invasioninhibitory response in individuals immune to malaria. J Exp Med 2001, 193:1403-1412.
3.
Bojang KA, Milligan PJ, Pinder M, Vigneron L, Alloueche A, Kester KE, Ballou WR, Conway DJ, Reece WH, Gothard P et al.: Efficacy of RTS,S/AS02 malaria vaccine against Plasmodium falciparum infection in semi-immune adult men in The Gambia: a randomized trial. Lancet 2001, 358:1927-1934.
4.
Kester KE, McKinney DA, Tornieporth N, Ockenhouse CF, Heppner DG, Hall T, Krzych U, Delchambre M, Voss G, Dowler MG et al.: Efficacy of recombinant circumsporozoite protein vaccine regimens against experimental Plasmodium falciparum malaria. J Infect Dis 2001, 183:640-647.
5.
Birkett A, Lyons K, Schmidt A, Boyd D, Oliveira GA, Siddique A, Nussenzweig R, Calvo-Calle JM, Nardin E: A modified hepatitis B virus core particle containing multiple epitopes of the Plasmodium falciparum circumsporozoite protein provides a highly immunogenic malaria vaccine in pre-clinical analyses in rodent and primate hosts. Infect Immun 2002, 70:6860-6870.
6.
Campos-Neto A, Porrozzi R, Greeson K, Coler RN, Webb JR, Seiky YA, Reed SG, Grimaldi G Jr: Protection against cutaneous leishmaniasis induced by recombinant antigens in murine and nonhuman primate models of the human disease. Infect Immun 2001, 69:4103-4108.
7.
Campos-Neto A, Webb JR, Greeson K, Coler RN, Skeiky YA, Reed SG: Vaccination with plasmid DNA encoding TSA/LmSTI1 leishmanial fusion proteins confers protection against Leishmania major infection in susceptible BALB/c mice. Infect Immun 2002, 70:2828-2836.
8.
Skeiky YA, Coler RN, Brannon M, Stromberg E, Greeson K, Crane RT, Campos-Neto A, Reed SG: Protective efficacy of a tandemly linked, multi-subunit recombinant leishmanial vaccine (Leish-111f) formulated in MPL adjuvant. Vaccine 2002, 20:3292-3303.
Conclusions Not all antigens are protective, but several vaccine candidates for most complex microorganisms exist. A plethora of well-defined recombinant protein vaccine candidates against intracellular organisms, such as M. tuberculosis, Plasmodium and Leishmania have been described and characterized over the past several years. Modern adjuvants that modulate T-cell mediated immunity (both CD4þ and CD8þ) to these antigens have been, and continue to be, developed. The recent discovery of protective cell-surfaceassociated protein antigens for extracellular organisms, such as S. pneumoniae and N. meningitidis, will soon lead to the generation of recombinant protein vaccine candidates that will replace the current complex mixture of illcharacterized capsular polysaccharides. Modern or classical adjuvants will help to promote both antibody-mediated immunity and long-lasting immunological memory (which does not exist for the current polysaccharide vaccines). These significant advances in both antigen characterization and delivery/adjuvant technology greatly improve the prospect that vaccines for these and other important infectious diseases will be possible in the near future. The shift from animal models to clinical development has finally www.current-opinion.com
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9.
Coler RN, Skeiky YA, Bernards K, Greeson K, Carter D, Cornellison CD, Modabber F, Campos-Neto A, Reed SG: Immunization with a polyprotein vaccine consisting of the T-Cell antigens thiol-specific antioxidant, Leishmania major stress-inducible protein 1, and Leishmania elongation initiation factor protects against leishmaniasis. Infect Immun 2002, 70:4215-4225. Using MPL1, a potent adjuvant acceptable for human use, a polyprotein consisting of three important leishmania antigens protected highly susceptible Balb/c mice with as little as 2mg protein. Current Opinion in Immunology 2003, 15:456–460
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10. Badaro R, Lobo I, Nakatani M, Muinos A, Netto EM, Coler RN, Reed SG: Successful use of a defined antigen/GM-CSF adjuvant vaccine to treat mucosal leishmaniasis refractory to antimony: a case report. Braz J Infect Dis 2001, 5:223-232.
22. Weinrich OA, van Pinxteren LA, Meng OL, Birk RP, Andersen P: Protection of mice with a tuberculosis subunit vaccine based on a fusion protein of antigen 85b and esat-6. Infect Immun 2001, 69:2773-2778.
11. Borges MM, Campos-Neto A, Sleath P, Grabstein KH, Morrissey PJ, Skeiky YA, Reed SG: Potent stimulation of the innate immune system by a Leishmania brasiliensis recombinant protein. Infect Immun 2001, 69:5270-5277.
23. Reed SG, Alderson MR, Dalemans W, Lobet Y, Skeiky YAW: Prospects for a better vaccine against tuberculosis. Tuberculosis 2003, in press. [Au: do you have any further details of this paper yet?]
12. Valenzuela JG, Belkaid Y, Garfield MK, Mendez S, Kamhawi S, Rowton ED, Sacks DL, Ribeiro JM: Toward a defined anti-Leishmania vaccine targeting vector antigens: characterization of a protective salivary protein. J Exp Med 2001, 194:331-342.
24. Comanducci M, Bambini S, Brunelli B, Adu-Bobie J, Arico B, Capecchi B, Giuliani MM, Masignani V, Santini L, Savino S et al.: NadA, a novel vaccine candidate of Neisseria meningitidis. J Exp Med 2002, 195:1445-1454. This paper describes an interesting cell-surface protein in N. meningitidis, which is present in 52 out of 53 virulent strains of the bacteria. This molecule induces strong bactericidal and protective antibodies in infant rat model of the infection, thus suggesting that this protein may be a novel vaccine candidate to control meningococcal diseases.
13. Reed SG: Leishmaniasis vaccination: targeting the source of infection. J Exp Med 2001, 194:F7-F9. 14. Behar SM, Dascher CC, Grusby MJ, Wang CR, Brenner MB: Susceptibility of mice deficient in CD1D or TAP1 to infection with Mycobacterium tuberculosis. J Exp Med 1999, 189:1973-1980. 15. Heinzel AS, Grotzke JE, Lines RA, Lewinsohn DA, McNabb AL, Streblow DN, Braud VM, Grieser HJ, Belisle JT, Lewinsohn DM: HLA-E-dependent presentation of Mtb-derived antigen to human CD8þ T cells. J Exp Med 2002, 196:1473-1481. 16. Sambandamurthy VK, Wang X, Chen B, Russell RG, Derrick S, Collins FM, Morris SL, Jacobs WR Jr: A pantothenate auxotroph of Mycobacterium tuberculosis is highly attenuated and protects mice against tuberculosis. Nat Med 2002, 8:1171-1174. 17. Hondalus MK, Bardarov S, Russell R, Chan J, Jacobs WR Jr, Bloom BR: Attenuation of and protection induced by a leucine auxotroph of Mycobacterium tuberculosis. Infect Immun 2000, 68:2888-2898. 18. Jackson M, Phalen SW, Lagranderie M, Ensergueix D, Chavarot P, Marchal G, McMurray DN, Gicquel B, Guilhot C: Persistence and protective efficacy of a Mycobacterium tuberculosis auxotroph vaccine. Infect Immun 1999, 67:2867-2873. 19. Smith DA, Parish T, Stoker NG, Bancroft GJ: Characterization of auxotrophic mutants of Mycobacterium tuberculosis and their potential as vaccine candidates. Infect Immun 2001, 69:1142-1150.
25. Etz H, Minh DB, Henics T, Dryla A, Winkler B, Triska C, Boyd AP, Sollner J, Schmidt W, von Ahsen U et al.: Identification of in vivo expressed vaccine candidate antigens from Staphylococcus aureus. Proc Natl Acad Sci USA 2002, 99:6573-6578. This paper describes a rapid and efficient antigen discovery strategy for the identification and cloning of genes encoding microbial cell surface proteins. Such an approach is a major advance in the development of vaccines against extracellular microorganisms. 26. Tettelin H, Nelson KE, Paulsen IT, Eisen JA, Read TD, Peterson S, Heidelberg J, DeBoy RT, Haft DH, Dodson RJ et al.: Complete genome sequence of a virulent isolate of Streptococcus pneumoniae. Science 2001, 293:498-506. 27. Parkhill J, Achtman M, James KD, Bentley SD, Churcher C, Klee SR, Morelli G, Basham D, Brown D, Chillingworth T et al.: Complete DNA sequence of a serogroup A strain of Neisseria meningitidis Z2491. Nature 2000, 404:502-506. 28. Tettelin H, Saunders NJ, Heidelberg J, Jeffries AC, Nelson KE, Eisen JA, Ketchum KA, Hood DW, Peden JF, Dodson RJ et al.: Complete genome sequence of Neisseria meningitidis serogroup B strain MC58. Science 2000, 287:1809-1815. 29. Pym AS, Brodin P, Majlessi L, Brosch R, Demangel C, Williams A, Griffiths KE, Marchal G, Leclerc C, Cole ST: Recombinant BCG exporting ESAT-6 confers enhanced protection against tuberculosis. Nat Med 2003, 9:533-539.
20. Rhee EG, Mendez S, Shah JA, Wu CY, Kirman JR, Turon TN, Davey DF, Davis H, Klinman DM, Coler RN et al.: Vaccination with heat-killed leishmania antigen or recombinant leishmanial protein and CpG oligodeoxynucleotides induces long-term memory CD4þ and CD8þ T cell responses and protection against Leishmania major infection. J Exp Med 2002, 195:1565-1573.
30. Bao L, Chen W, Zhang H, Wang X: Virulence, immunogenicity, and protective efficacy of two recombinant Mycobacterium bovis Bacillus Calmette-Guerin strains expressing the antigen ESAT-6 from Mycobacterium tuberculosis. Infect Immun 2003, 71:1656-1661.
21. Gierynska M, Kumaraguru U, Eo SK, Lee S, Krieg A, Rouse BT: Induction of CD8 T-cell-specific systemic and mucosal immunity against herpes simplex virus with CpG-peptide complexes. J Virol 2002, 76:6568-6576.
31. Horwitz MA, Harth G: A new vaccine against tuberculosis affords greater survival after challenge than the current vaccine in the guinea pig model of pulmonary tuberculosis. Infect Immun 2003, 71:1672-1679.
Current Opinion in Immunology 2003, 15:456–460
www.current-opinion.com
Vaccines for parasitic and bacterial diseases Steven G Reedy and Antonio Campos-Netoz The first decade of the millennium should mark the beginning of a new era in vaccine development, reaping the rewards of advances in genome characterization, antigen identification, understanding the molecular bases of protective immune responses, and adjuvant design and development. Advances in all of these areas have culminated in vaccine candidates entering clinical testing. These include vaccines against two of humankind’s oldest and deadliest diseases, tuberculosis and malaria. Several vaccine candidates for each of these diseases will be tested in humans during the next few years. A candidate vaccine for leishmaniasis, an infection that has taught us much about T-cell regulation of protection and disease in animal models, has been developed and is now in the clinic. There are indications both in animal models and in patients that vaccines may be used not only to protect but also to treat leishmania infections. Addresses Infectious Disease Research Institute, 1124 Columbia Street, Suite 600, Seattle, WA 98104, USA y Corixa Corporation, 1124 Columbia Street, Suite 200, Seattle, WA 98104, USA; and, University of Washington, Department of Pathobiology, Box 357238, Seattle, WA 98195, USA e-mail: [email protected] z e-mail: [email protected]
Current Opinion in Immunology 2003, 15:456–460 This review comes from a themed issue on Host–pathogen interactions Edited by Robert L Modlin and Peter Doherty 0952-7915/$ – see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0952-7915(03)00069-4
Abbreviations BCG bacillus Calmette-Gue´rin IFN interferon MPL monophosphoryl lipid A MPL-SE MPL stable emulsion
Introduction The pace of vaccine development for complex organisms has been accelerated in the past decade because of advances in antigen discovery including genome sequencing, increased understanding of immune mechanisms of protection and, perhaps most importantly, the development of safe and effective vaccine adjuvants and delivery platforms. Only a few years ago it was thought that protective T-cell responses to intracellular pathogens could be achieved only by immunization with live vaccines or by using adjuvants deemed unacceptable for humans because Current Opinion in Immunology 2003, 15:456–460
of reactogenicity. Over the past several years it has become evident that a new generation of vaccines is feasible. Most of these vaccines will consist of defined antigens, and many will involve new adjuvants or delivery technologies capable of stimulating a broad range of immune responses. In this brief review, we will focus on a few infectious agents for which significant advances in vaccine development have been made very recently.
Malaria Significant progress has been made on moving promising candidates into development for clinical trials. One candidate that has moved into the clinic is the 19 kDa merozoite surface antigen (MSP-1) of Plasmodium falciparum. This protein was tested as an alum-adsorbed vaccine in a Phase I trial of normal volunteers, in whom induction of a range of cytokines was detected [1]. The importance of MSP-1 was further documented in a study demonstrating that antibodies to this surface protein were important in blocking parasite invasion of erythrocytes in individuals immune to malaria [2]. One of the more interesting advances in malaria vaccine development is the use of parasite antigens coupled to particulate viral proteins as a way to induce potent immune responses. The RTS,S vaccine developed by GlaxoSmithKline Biologicals (Rixensart, Belgium) and collaborators includes the P. falciparum circumsporozoite (CS) protein expressed in a hepatitis B surface antigen particle to increase immunogenicity. The vaccine is delivered in ASO2, an adjuvant that contains monophosphoryl lipid A (MPL1; Corixa Corporation, Hamilton, MT, USA) and QS-21. The vaccine was previously shown to induce partial protection in human challenge experiments and more recently has been tested in African field trials where significant, although not long-lasting, protection was reported [3,4]. Another approach to induce potent immune responses to the CS protein was used by Birkett et al. [5], who expressed B- and T-cell epitopes of this protein in a hepatitis B core protein. This construct, termed ICC-1132, was highly immunogenic when formulated with alum or in Montanide ISA 720. This approach may be applicable for antigens for which epitopes of limited size can be inserted into the viral core protein. The ICC-1132 vaccine is scheduled for clinical trials soon.
Leishmaniasis The intracellular parasites belonging to the Leishmania complex have been used for decades in vaccine models for diseases requiring potent T-cell immunity. A few antigens that induce high degrees of protection have been identified, but until recently no practical adjuvants www.current-opinion.com
Vaccines for parasites and bacteria Reed and Campos-Neto
or delivery systems were available to move vaccine candidates into the clinic. A mixture of three antigens (thiol-specific antioxidant [TSA], Leishmania major stressinducible protein-1 [LmSTI-1] and Leishmania elongation initiation factor [LeIF]) has been found to induce protection in mouse and monkey models when delivered in IL-12 or as DNA [6,7]. Other adjuvants have been evaluated in the BALB/c mouse model, including CpG and MPL1 stable emulsion (MPL-SE). The three vaccine antigens have been produced as a single fusion protein, Leish-111f, which conferred excellent protection as DNA or protein [8,9]. A candidate vaccine consisting of Leish-111f formulated in MPL-SE entered Phase I clinical testing in normal volunteers in January 2003. The three antigens that make up Leish-111f have been used to develop an immunotherapeutic strategy to treat patients with refractory mucosal leishmaniasis [10]. This report follows previous studies in which crude parasite preparations, either alone or with the live attenuated strain of Mycobacterium bovis — bacillus Calmette-Gue´ rin (BCG), were administered to patients with mucosal leishmaniasis. The earlier studies with crude vaccine preparations, and later with defined antigens, have demonstrated that vaccines can be used to alter the course of an ongoing disease characterized not by an anergic state, but rather by hyperreactivity to parasite antigens. In a recent study, we examined T-cell responses to parasite antigens pre- and post-immunization with the recombinant proteins that make up Leish-111f. Preliminary data from our laboratory indicates that, although patient peripheral blood mononuclear cells (PBMCs) proliferated strongly in response to parasite lysate before immunization, the recombinant antigens elicited much weaker proliferative responses in most cases. Not surprisingly, IFN-g production in response to the recombinant antigens increased following immunization. The most significant finding, however, was a marked decrease in IL-5 production in response to parasite lysate following immunization with the recombinant proteins. Although this response was high before immunization, it decreased to background levels after the third vaccine dose. The decrease in IL-5 production correlated with improvement in, or resolution of, clinical signs and symptoms. These observations support the concept that a T-cell vaccine can be used to alter an ongoing immune response and consequently to treat disease. Understanding the properties of a vaccine that are responsible for this will help in the design of effective immunotherapeutics. One plausible mechanism is the induction of IL-12 by LeIF, one of the components of the vaccine. LeIF stimulates potent innate immune responses, and should perhaps be considered as an important component of an effective vaccine or immunotherapeutic [11]. Another exciting and controversial approach is vaccinating against vector-borne diseases, as reported by Sacks, www.current-opinion.com
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Ribeiro and colleagues [12]. To achieve protection against leishmaniasis, this group immunized mice with naked DNA encoding a protein from the salivary gland from the sandfly vector instead of with proteins from the parasites [12]. Solid protection accompanied by high IFN-g production was observed after natural challenge of the mice using infected sandflies. Protection was presumably conferred in part by an inflammatory process mediated by immune recognition of the fly components. The implications of this approach for vaccine development have been discussed elsewhere [13].
Mycobacterium tuberculosis Approximately one-third of the world’s population is infected with M. tuberculosis and it is estimated that this disease causes three million deaths each year. Despite the effective treatment of tuberculosis with a combination of three antibiotics the treatment is long, resulting in inadequate compliancy and leading to the development of several multidrug-resistant strains of M. tuberculosis. Compounded with the emergence of the M. tuberculosis-HIV co-infection epidemic in both developing and industrialized countries, this underscores the need for an effective vaccine against the disease. The current and only available vaccine, BCG, although efficacious in preventing the less common yet severe forms of the disease in young children (meningitis and systemic disease) has no effect against adult pulmonary tuberculosis, the most common form of the disease. A serious complicating factor is our lack of a clear understanding of the mechanism of protection in tuberculosis. It is clear, however, that the CD4þ T-cell response plays an important role in immunity. CD8þ T cells may also participate in resistance but in a more restricted manner, perhaps keeping the latent phase of the infectious process in check. The strong T-cell response to CD1-associated non-protein antigens of M. tuberculosis observed in many sensitized individuals, though intriguing and unique, has not proven to be involved in the immune defense mechanisms against tuberculosis [14]. More recently, another unique system has been described for T-cell recognition of M. tuberculosis antigens [15]. CD8þ T cells from people with latent tuberculosis infection recognize pathogen antigens in complex with HLA-E, as well as in complex with the more conventional HLA-A, -B, or -C MHC class I molecules. The implication of these findings for resistance to tuberculosis has not yet been studied. Over the past decade several investigators have concentrated major efforts in the development of a better vaccine than BCG. A number of different approaches have been pursued. Most have focused on the development of a recombinant subunit vaccine (protein or plasmid DNA), while others have concentrated either on the improvement of the BCG vaccination or in the development of viable attenuated M. tuberculosis. The molecular Current Opinion in Immunology 2003, 15:456–460
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manipulation of BCG, by insertion of either mammalian genes encoding cytokines (aiming to enhance or modulate the immune response induced by the bacteria) or M. tuberculosis genes encoding proteins of the tubercle bacilli, resulted in mycobacterial variants that at best, marginally augmented the protective efficacy of the original BCG strain. However, some success has recently been reported with recombinant BCG strains (see also Update). In addition, genetic manipulation of virulent M. tuberculosis has generated interesting auxotrophic mutants with low virulence and protective efficacy similar to that induced by BCG in mice challenged with the virulent bacilli [16–19]. Despite the fact that the protection induced by these auxotrophic mutants has not surpassed BCG, this approach resulted in a highly protective vaccine. Viable attenuated microorganisms are the components of highly efficient vaccines against several human infectious diseases, including smallpox, polio, measles, mumps and leishmaniasis. Attenuation of virulence of the microorganisms present in these vaccines resulted either from an accident of nature (e.g. smallpox) or from empirical manipulations of the microbe cultures. The idea and feasibility of using rational and modern molecular genetic approaches to engineer a non-virulent M. tuberculosis strain that preserves most of the antigenic repertoire of the virulent strain is highly attractive and has the potential to generate a viable anti-tuberculosis vaccine that is superior to BCG. Much progress has also been made in the development of an anti-tuberculosis recombinant subunit vaccine. In particular, bacterial plasmid DNAs containing M. tuberculosis genes have been extensively tested over the past ten years. However, the initial euphoria associated with this method of antigen delivery has been hampered by an inability to reproduce the exciting original results beyond the murine model of tuberculosis. A plethora of publications describing the protection of mice, induced by many different M. tuberculosis DNAs, could not be reproduced in either the guinea pig or monkey models. Despite several attempts at optimizing codon usage and alternating priming/boosting with either BCG or adenovirus carrying the M. tuberculosis genes, there was little evidence of improvement. More encouraging, however, are the results obtained with conventional protein immunization formulated with newer adjuvants capable of both amplifying and modulating the immune response to the T helper 1 (Th1) phenotype. Examples are the MPL1 and a series of adjuvants containing MPL1 in association with CpG oligonucleotides (AS02 adjuvants from GlaxoSmithKline). Importantly, these adjuvants are safe for human use [3]. Somewhat unexpectedly, these approaches promote the emergence of CD8þ-specific T-cell responses to the protein antigen [20,21]. As mentioned above, both Th1 CD4þ T cells and CD8þ T cells are important Current Opinion in Immunology 2003, 15:456–460
mediators of immunity against tuberculosis. Also encouraging are the results obtained with recombinant polyproteins of M. tuberculosis. Such approaches (i.e. fusing the genes encoding proteins that induce partial protection) result in single larger molecules that preserve both the immunogenicity and protective efficacy of each individual component of the polyprotein [22]. Excellent shortand long-term protection has been obtained using such molecules formulated with the adjuvant AS02 (GlaxoSmithKline) in mice, guinea pig and monkey models of tuberculosis [23]. In conclusion, the development of both interesting auxotrophic mutants of M. tuberculosis (viable attenuated vaccine) and recombinant polyproteins of the tubercle bacilli associated with unique adjuvants (subunit vaccine) should ultimately result in a more efficacious vaccine than the current BCG.
Streptococcus pneumoniae and Neisseria meningitidis Streptococcus pneumoniae is the leading etiological agent of bacterial pneumonia, meningitis and otitis media in both adults and children. Approximately one million children under the age of five die every year from the pneumonia caused by S. pneumoniae. Neisseria meningitidis causes endemic and epidemic bacterial meningitis. The serious and frequent diseases caused by these organisms occur in both developed and developing nations. In contrast to M. tuberculosis, S. pneumoniae and N. meningitidis are extracellular infectious agents, and immunity to them is primarily mediated by specific antibodies to the bacterial surface antigens. Both organisms produce extensive polysaccharide capsules, which are the antigenic target of existing vaccines against these bacteria. However, one serious complicating factor with these vaccines is the immense antigenic heterogeneity of the capsular polysaccharide of both organisms. For example, there are at least 23 common serotypes of S. pneumoniae that cause disease in humans. Similarly, N. meningitidis can be segregated into at least 13 serogroups. The available vaccines for use in the United States against S. pneumoniae contain a mixture of 25mg of polysaccharide of each of the 23 prevalent serotypes. Currently available vaccines against N. meningitidis include a quadrivalent product containing the polysaccharides of groups A, C, Y and W-135. No vaccine is available for group B because, for unknown reasons, this polysaccharide is not immunogenic in humans. Another serious complicating factor with these vaccines is the polysaccharide nature of their antigens. Because the immune response to such antigens is T-cell independent, immunological memory is not elicited and there is thus no boosting effect with repeated immunizations. The alternative approach of conjugating a microbial protein carrier to the polysaccharide has slightly improved the efficacy of the anti-polysaccharide C group of N. meningitidis. Thus far, no convincing data exists to show improvement in either antibody titers or the induction of memory to S. pneumoniae polysaccharide. www.current-opinion.com
Vaccines for parasites and bacteria Reed and Campos-Neto
Although the concept that ideal vaccines against microbes for which immunity is antibody mediated should include surface proteins that are conserved across all the serotypes/serogroups is not new, the identification and characterization of such molecules has been difficult. Notwithstanding, one such molecule has recently been described [24]. More importantly, another recent report describes an interesting general approach for the identification, screening and cloning of the genes encoding bacterial cell-surface peptides [25]. A Staphylococcus aureus genomic DNA library was initially generated using short DNA fragments ranging from 150 to 300bps. To facilitate cell-surface expression, the DNA fragments were digested with restriction enzymes and fused with the genes encoding one of the two outer membrane proteins LamB and FhuA. The use of high-titer human sera from patients suffering from moderate to severe S. aureus infections, in combination with magnetic cell sorting and an opsonophagocytosis assay to screen the library, resulted in the cloning and sequencing of several genes. Alignment of these sequences with the S. aureus genome revealed that several of the corresponding fulllength genes encoded proteins that were either located on the surface of the bacterium or secreted. An antibody specific for one such peptide revealed its presence on the cell surface of S. aureus and, more importantly, facilitated the phagocytosis of these organisms by human polymorphonuclear cells. This approach should therefore be of great value for the identification of protein vaccine candidates for pathogens, where immunity is antibody mediated and the complete genome sequence has been determined, as in the case of S. pneumonia and N. meningitidis [26–28].
begun, so that we can now begin to learn how to build successful vaccines for these challenging diseases.
Update Recent work has shown that recombinant BCG expressing ESAT-6 or the 30 kDa secretory protein confers enhanced protection against challenge with virulent M. tuberculosis [29–31].
Acknowledgements This work supported in part by National Institutes of Health grants AI-25038, AI-43528, AI-44373, and AI-49505.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
Lee EA, Palmer DR, Flanagan KL, Reece WH, Odhiambo K, Marsh K, Pinder M, Gravenor MB, Keitel WA, Kester KE et al.: Induction of T helper type 1 and 2 responses to 19-kilodalton merozoite surface protein 1 in vaccinated healthy volunteers and adults naturally exposed to malaria. Infect Immun 2002, 70:1417-1421.
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O’Donnell RA, Koning-Ward TF, Burt RA, Bockarie M, Reeder JC, Cowman AF, Crabb BS: Antibodies against merozoite surface protein (MSP)-1(19) are a major component of the invasioninhibitory response in individuals immune to malaria. J Exp Med 2001, 193:1403-1412.
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Bojang KA, Milligan PJ, Pinder M, Vigneron L, Alloueche A, Kester KE, Ballou WR, Conway DJ, Reece WH, Gothard P et al.: Efficacy of RTS,S/AS02 malaria vaccine against Plasmodium falciparum infection in semi-immune adult men in The Gambia: a randomized trial. Lancet 2001, 358:1927-1934.
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Kester KE, McKinney DA, Tornieporth N, Ockenhouse CF, Heppner DG, Hall T, Krzych U, Delchambre M, Voss G, Dowler MG et al.: Efficacy of recombinant circumsporozoite protein vaccine regimens against experimental Plasmodium falciparum malaria. J Infect Dis 2001, 183:640-647.
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Birkett A, Lyons K, Schmidt A, Boyd D, Oliveira GA, Siddique A, Nussenzweig R, Calvo-Calle JM, Nardin E: A modified hepatitis B virus core particle containing multiple epitopes of the Plasmodium falciparum circumsporozoite protein provides a highly immunogenic malaria vaccine in pre-clinical analyses in rodent and primate hosts. Infect Immun 2002, 70:6860-6870.
6.
Campos-Neto A, Porrozzi R, Greeson K, Coler RN, Webb JR, Seiky YA, Reed SG, Grimaldi G Jr: Protection against cutaneous leishmaniasis induced by recombinant antigens in murine and nonhuman primate models of the human disease. Infect Immun 2001, 69:4103-4108.
7.
Campos-Neto A, Webb JR, Greeson K, Coler RN, Skeiky YA, Reed SG: Vaccination with plasmid DNA encoding TSA/LmSTI1 leishmanial fusion proteins confers protection against Leishmania major infection in susceptible BALB/c mice. Infect Immun 2002, 70:2828-2836.
8.
Skeiky YA, Coler RN, Brannon M, Stromberg E, Greeson K, Crane RT, Campos-Neto A, Reed SG: Protective efficacy of a tandemly linked, multi-subunit recombinant leishmanial vaccine (Leish-111f) formulated in MPL adjuvant. Vaccine 2002, 20:3292-3303.
Conclusions Not all antigens are protective, but several vaccine candidates for most complex microorganisms exist. A plethora of well-defined recombinant protein vaccine candidates against intracellular organisms, such as M. tuberculosis, Plasmodium and Leishmania have been described and characterized over the past several years. Modern adjuvants that modulate T-cell mediated immunity (both CD4þ and CD8þ) to these antigens have been, and continue to be, developed. The recent discovery of protective cell-surfaceassociated protein antigens for extracellular organisms, such as S. pneumoniae and N. meningitidis, will soon lead to the generation of recombinant protein vaccine candidates that will replace the current complex mixture of illcharacterized capsular polysaccharides. Modern or classical adjuvants will help to promote both antibody-mediated immunity and long-lasting immunological memory (which does not exist for the current polysaccharide vaccines). These significant advances in both antigen characterization and delivery/adjuvant technology greatly improve the prospect that vaccines for these and other important infectious diseases will be possible in the near future. The shift from animal models to clinical development has finally www.current-opinion.com
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9.
Coler RN, Skeiky YA, Bernards K, Greeson K, Carter D, Cornellison CD, Modabber F, Campos-Neto A, Reed SG: Immunization with a polyprotein vaccine consisting of the T-Cell antigens thiol-specific antioxidant, Leishmania major stress-inducible protein 1, and Leishmania elongation initiation factor protects against leishmaniasis. Infect Immun 2002, 70:4215-4225. Using MPL1, a potent adjuvant acceptable for human use, a polyprotein consisting of three important leishmania antigens protected highly susceptible Balb/c mice with as little as 2mg protein. Current Opinion in Immunology 2003, 15:456–460
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10. Badaro R, Lobo I, Nakatani M, Muinos A, Netto EM, Coler RN, Reed SG: Successful use of a defined antigen/GM-CSF adjuvant vaccine to treat mucosal leishmaniasis refractory to antimony: a case report. Braz J Infect Dis 2001, 5:223-232.
22. Weinrich OA, van Pinxteren LA, Meng OL, Birk RP, Andersen P: Protection of mice with a tuberculosis subunit vaccine based on a fusion protein of antigen 85b and esat-6. Infect Immun 2001, 69:2773-2778.
11. Borges MM, Campos-Neto A, Sleath P, Grabstein KH, Morrissey PJ, Skeiky YA, Reed SG: Potent stimulation of the innate immune system by a Leishmania brasiliensis recombinant protein. Infect Immun 2001, 69:5270-5277.
23. Reed SG, Alderson MR, Dalemans W, Lobet Y, Skeiky YAW: Prospects for a better vaccine against tuberculosis. Tuberculosis 2003, in press. [Au: do you have any further details of this paper yet?]
12. Valenzuela JG, Belkaid Y, Garfield MK, Mendez S, Kamhawi S, Rowton ED, Sacks DL, Ribeiro JM: Toward a defined anti-Leishmania vaccine targeting vector antigens: characterization of a protective salivary protein. J Exp Med 2001, 194:331-342.
24. Comanducci M, Bambini S, Brunelli B, Adu-Bobie J, Arico B, Capecchi B, Giuliani MM, Masignani V, Santini L, Savino S et al.: NadA, a novel vaccine candidate of Neisseria meningitidis. J Exp Med 2002, 195:1445-1454. This paper describes an interesting cell-surface protein in N. meningitidis, which is present in 52 out of 53 virulent strains of the bacteria. This molecule induces strong bactericidal and protective antibodies in infant rat model of the infection, thus suggesting that this protein may be a novel vaccine candidate to control meningococcal diseases.
13. Reed SG: Leishmaniasis vaccination: targeting the source of infection. J Exp Med 2001, 194:F7-F9. 14. Behar SM, Dascher CC, Grusby MJ, Wang CR, Brenner MB: Susceptibility of mice deficient in CD1D or TAP1 to infection with Mycobacterium tuberculosis. J Exp Med 1999, 189:1973-1980. 15. Heinzel AS, Grotzke JE, Lines RA, Lewinsohn DA, McNabb AL, Streblow DN, Braud VM, Grieser HJ, Belisle JT, Lewinsohn DM: HLA-E-dependent presentation of Mtb-derived antigen to human CD8þ T cells. J Exp Med 2002, 196:1473-1481. 16. Sambandamurthy VK, Wang X, Chen B, Russell RG, Derrick S, Collins FM, Morris SL, Jacobs WR Jr: A pantothenate auxotroph of Mycobacterium tuberculosis is highly attenuated and protects mice against tuberculosis. Nat Med 2002, 8:1171-1174. 17. Hondalus MK, Bardarov S, Russell R, Chan J, Jacobs WR Jr, Bloom BR: Attenuation of and protection induced by a leucine auxotroph of Mycobacterium tuberculosis. Infect Immun 2000, 68:2888-2898. 18. Jackson M, Phalen SW, Lagranderie M, Ensergueix D, Chavarot P, Marchal G, McMurray DN, Gicquel B, Guilhot C: Persistence and protective efficacy of a Mycobacterium tuberculosis auxotroph vaccine. Infect Immun 1999, 67:2867-2873. 19. Smith DA, Parish T, Stoker NG, Bancroft GJ: Characterization of auxotrophic mutants of Mycobacterium tuberculosis and their potential as vaccine candidates. Infect Immun 2001, 69:1142-1150.
25. Etz H, Minh DB, Henics T, Dryla A, Winkler B, Triska C, Boyd AP, Sollner J, Schmidt W, von Ahsen U et al.: Identification of in vivo expressed vaccine candidate antigens from Staphylococcus aureus. Proc Natl Acad Sci USA 2002, 99:6573-6578. This paper describes a rapid and efficient antigen discovery strategy for the identification and cloning of genes encoding microbial cell surface proteins. Such an approach is a major advance in the development of vaccines against extracellular microorganisms. 26. Tettelin H, Nelson KE, Paulsen IT, Eisen JA, Read TD, Peterson S, Heidelberg J, DeBoy RT, Haft DH, Dodson RJ et al.: Complete genome sequence of a virulent isolate of Streptococcus pneumoniae. Science 2001, 293:498-506. 27. Parkhill J, Achtman M, James KD, Bentley SD, Churcher C, Klee SR, Morelli G, Basham D, Brown D, Chillingworth T et al.: Complete DNA sequence of a serogroup A strain of Neisseria meningitidis Z2491. Nature 2000, 404:502-506. 28. Tettelin H, Saunders NJ, Heidelberg J, Jeffries AC, Nelson KE, Eisen JA, Ketchum KA, Hood DW, Peden JF, Dodson RJ et al.: Complete genome sequence of Neisseria meningitidis serogroup B strain MC58. Science 2000, 287:1809-1815. 29. Pym AS, Brodin P, Majlessi L, Brosch R, Demangel C, Williams A, Griffiths KE, Marchal G, Leclerc C, Cole ST: Recombinant BCG exporting ESAT-6 confers enhanced protection against tuberculosis. Nat Med 2003, 9:533-539.
20. Rhee EG, Mendez S, Shah JA, Wu CY, Kirman JR, Turon TN, Davey DF, Davis H, Klinman DM, Coler RN et al.: Vaccination with heat-killed leishmania antigen or recombinant leishmanial protein and CpG oligodeoxynucleotides induces long-term memory CD4þ and CD8þ T cell responses and protection against Leishmania major infection. J Exp Med 2002, 195:1565-1573.
30. Bao L, Chen W, Zhang H, Wang X: Virulence, immunogenicity, and protective efficacy of two recombinant Mycobacterium bovis Bacillus Calmette-Guerin strains expressing the antigen ESAT-6 from Mycobacterium tuberculosis. Infect Immun 2003, 71:1656-1661.
21. Gierynska M, Kumaraguru U, Eo SK, Lee S, Krieg A, Rouse BT: Induction of CD8 T-cell-specific systemic and mucosal immunity against herpes simplex virus with CpG-peptide complexes. J Virol 2002, 76:6568-6576.
31. Horwitz MA, Harth G: A new vaccine against tuberculosis affords greater survival after challenge than the current vaccine in the guinea pig model of pulmonary tuberculosis. Infect Immun 2003, 71:1672-1679.
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