Factors influencing the immune response to foreign antigen expressed in recombinant BCG vaccines

Vaccine 23 (2005) 1209–1224

Review

Factors influencing the immune response to foreign antigen expressed in recombinant BCG vaccines Maureen Dennehya,∗ , Anna-Lise Williamsonb a The Biovac Institute, Private Bag X3, Pinelands, 7430 Cape Town, South Africa Division of Virology, Institute of Infectious Diseases and Molecular Medicine, Faculty of Health Sciences, University of Cape Town and National Health Laboratory Services, Anzio Road, Observatory, Cape Town 7925, South Africa b

Received 21 May 2004; accepted 26 August 2004 Available online 7 October 2004

Abstract A wide range of recombinant BCG vaccine candidates containing foreign viral, bacterial, parasite or immunomodulatory genetic material have been developed and evaluated, primarily in animal models, for immune response to the foreign antigen. This review considers some of the factors that may influence the immunogenicity of these vaccines. The influence of levels and timing of expression of the foreign antigen and the use of targeting sequences are considered in the first section. Genetic and functional stability of rBCG is reviewed in the second section. In the last section, the influence of dose and route of immunization, strain of BCG and the animal model used are discussed. © 2004 Elsevier Ltd. All rights reserved. Keywords: Recombinant; BCG; Mycobacterium bovis

Contents

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1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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2.

Shuttle vector features. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Expression of foreign antigen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1. Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2. Translation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.3. Levels of recombinant protein in rBCG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Antigen display . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. Secreted antigen. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2. Membrane-anchored protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.3. The influence of targeting on immune response: comparative rBCG studies . . . . . . . . . . . . . . . . . . . .

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3.

Stability of the rBCG vaccine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Episomal vectors: in vitro stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Episomal vectors: in vivo stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Episomal versus integrating vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Stability of expressed protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Corresponding author. Tel.: +27 21 5112266; fax: +27 21 5113962. E-mail address: [email protected] (M. Dennehy).

0264-410X/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2004.08.039

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4.

Preclinical testing considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Dose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Immunization route . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.1. In vivo establishment of rBCG after different routes of immunization . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.2. Immune response and immunization route . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. BCG strain and preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Animal model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Prior exposure of animals to BCG or foreign antigen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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5.

Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction In the literature documenting the design and testing of rBCG vaccine candidates containing foreign genetic material (see [1] for a review), a number of factors have been identified as important in influencing the immune response against the foreign antigen. These may be divided into: (i) Shuttle vector features, such as expression system and use of targeting sequences. This will determine the timing and amount of protein produced and the timing and route of its presentation to the immune system. (ii) Stability of the rBCG vaccine. This has emerged as a significant consideration in use of rBCG. (iii) Preclinical testing considerations. The dose and route of immunization have significant effects on the subsequent immune response. Immunization route influences the establishment and spreading of BCG and both the nature and persistence of the subsequent immune response. The test system, including both strain of BCG and animal model used, is also significant.

2. Shuttle vector features A large number of E. coli-mycobacterium shuttle vectors have been developed for the transfer of the foreign genes into BCG. Key features of these vectors have a large influence on the extent and nature of the immune response that could be elicited by recombinant BCG hosting them. Firstly, the expression system determines the level of production of the foreign protein. Promoter elements have received particular attention, as it is feasible to regulate the level of expression of a given gene as a function of promoter strength and the timing of expression according to promoter activity. The inserted, foreign DNA sequence may, itself, influence expression level. Secondly, leader sequences used to target the protein within the BCG cell may influence the level of production

[2] and do influence the timing and pathway of presentation of foreign antigen to the immune system. The BCG ␣ antigen protein and the M. tuberculosis 19 kDa lipoprotein, for example, when fused to the foreign protein, provide the signals for export and membrane-anchoring, respectively. They are also considered in this review along with the predicted influence of such targeting on the immune response and supporting evidence from the rBCG literature. Most of the currently-available E. coli-mycobacterial shuttle vectors are maintained in mycobacteria either episomally or by integrating into the mycobacterial genome. Episomal plasmids were developed by combining a region of the mycobacterial replicon of the M. fortuitum pAL5000 plasmid [3] with an E. coli cloning vector and a kanamycin-resistance gene [4]. These shuttle vectors replicate in mycobacteria at about five copies per genome [5]. Lee et al. [6] used the integration system of mycobacteriophage L5—the attachment region, attP, and integrase gene, int—to achieve attB site-specific integration of the vector into the mycobacterial genome. Since these vectors lack a mycobacterial origin of replication, they must integrate in order to persist. However, only a single copy can integrate into the genome. 2.1. Expression of foreign antigen 2.1.1. Transcription Promoters that are active in mycobacteria are required to drive expression of foreign genes in BCG. M. leprae- or BCGderived [5] heat shock protein (hsp) 60 and BCG- [5] or M. tuberculosis-derived hsp70 [7] promoters have been widely used in the available E. coli-mycobacterium shuttle vectors. Other promoters used successfully in the early shuttle vectors include those from the M. kansasii ␣ (alpha) antigen [8], the M. paratuberculosis IS900 open reading frame (ORF) 2 (pAN [9]), the M. tuberculosis 19 kDa antigen [10] and the M. fortuitum ␤ lactamase (pBlaF* [11]), as well as a compatible promoter from Streptomyces albus, GroES/EL1

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[12]. Transcriptional terminators derived from E. coli [5,13] are used in most vectors. The strength of the promoter determines rate of transcription initiation, the rate-limiting step in mRNA production [14]. Expression of foreign genes in mycobacteria can be modulated at the transcriptional level as a function of promoter strength, although, as yet, levels of control are not as reliable and reproducible as is possible in more established systems of bacterial genetic manipulation. Developments in the understanding of mycobacterial promoter structure and function [14] do now allow the use of a wider range of promoters in attempts to regulate the level of expression of the foreign antigen, an approach that is being pursued in our laboratory and others [15,16]. Further refinements in transcriptional regulation involve the use of inducible promoters, specifically activated when the host bacterium is stressed, either in culture or when it infects cells. This avoids metabolic load on the bacterium during routine in vitro growth and manipulation, but induces foreign antigen expression at times when an immune response is required. The heat shock promoters, for example, are upregulated during stress, although significant levels of constitutive expression can be detected in vitro in both E. coli and mycobacteria [17] as the heat shock proteins they control are essential under all growth conditions [5]. The M. leprae 18 kDa antigen promoter [17,18] and the M. tuberculosis mtrA promoter [19] are activated in vivo after phagocytosis. The relative levels and differential activation of expression from three promoters used in our laboratory (BCG hsp60, the M. leprae 18 kDa antigen and the M. tuberculosis mtrA promoters) are reviewed in more detail below. 2.1.1.1. The hsp60 promoter. The hsp60 protein belongs to a large family of heat shock proteins, induced at high levels under conditions of stress, which facilitate prompt adaptive responses to hostile environments. The mycobacterial hsp60 promoter, described by Stover et al. [5] and Dellagostin et al. [17], has two transcriptional start sites in BCG, tsA and tsB. Only tsA is recognized in M. smegmatis [17]. This promoter shows constitutive activity in mycobacterial cultures, but is strongly induced under conditions of stress in culture and, possibly, during infection of macrophages. In M. avium, Batoni et al. [20] demonstrated low activity during exponential growth in standing culture, but a considerable increase in activity during late exponential to late stationary growth phases. Dhandayuthapani et al. [21] showed in M. smegmatis that hsp60 promoter activity was higher than that of the 18 kDa antigen promoter. Protein yield results of Da Cruz et al. [22] confirm this. Similarly, in M. smegmatis and BCG, Dellagostin et al. [17] showed hsp60 promoter activity during late exponential phase was higher than that of the 18 kDa antigen promoter. Interestingly, promoter activity was demonstrated in E. coli in this study. Hsp60 promoter activity can also be induced in cultures by experimental stresses, such as heat or acid stress. Batoni et al. [20] reported significant induction in exponentially grow-

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ing cells by temperature shift (37–45 ◦ C, but not 42 ◦ C), but no significant induction by addition of peroxide. Stover et al. [5] also noted induction after heat shock or addition of acid, but not peroxide. Haeseleer et al. [23], on the other hand, could detect no change after heat or peroxide shock in culture. In vivo, Batoni et al. [20] noted hsp60 promoter induction within 3 h of BCG infection of murine macrophages. Enhanced activity persisted over 7 days. Zahrt and Deretic [24], on the other hand, did not note significant induction after macrophage infection by M. tuberculosis or BCG, although promoter activity was consistently high. The hsp60 promoter, therefore, supports relatively high levels of transcription, which may be advantageous in the design of an rBCG candidate, provided the levels are not so high as to induce chaos. However, it can be highly active during in vitro growth in both E. coli and mycobacteria. This may represent a disadvantage relative to the 18 kDa antigen and mtrA promoters, which have lower activity in vitro and are induced on infection of macrophages. They would be expected, therefore, to drive production of a foreign antigen in rBCG mostly at a time the antigen could be exposed to the immune system. 2.1.1.2. The M. leprae 18 kDa promoter. The 18 kDa protein is a major antigen of infection with M. leprae. It is related to the ␣ crystallins, a group of low molecular weight heat-shock proteins of unknown function [17]. Homologues are found in other mycobacteria [25]. The transcription start site, identified by Dellagostin et al. [17], has indicated likely −10 and −35 promoter sequences of the 18 kDa promoter. An undefined upstream element also appears to be involved in the regulation of expression, as consistently higher expression from the same site occurs both in vitro (in E. coli, M. smegmatis and BCG) and in BCG-infected macrophages if more upstream DNA (256 bp compared to 136 bp) is included in the expression system. Promoter activity has been described by Dellagostin et al. [17]. Measured at late exponential phase in shaken liquid culture, promoter activity was weak in M. smegmatis and BCG, but, surprisingly, relatively strong in E. coli. Expression of a homologue in M. habana was found to increase after heat shock, although this did not occur M. tuberculosis. In vivo activity within BCG-infected macrophages was always lower than that of the hsp60 promoter, although the ratio of activation on infection of macrophages was higher, showing induction. 2.1.1.3. The mtrA promoter. The M. tuberculosis mtrA protein is part of a two-component signal-transduction system (mtrA–mtrB; [24]). These systems are ubiquitous bacterial regulatory elements associated with environmental signal recognition and the induction of adaptive responses. Unusually for two-component signal-transduction systems, mtrA (but not mtrB) is essential for M. tuberculosis viability in vitro. It is not known whether it is also essential for the growth of BCG, although this is significant for its use to drive the expression of foreign genes in BCG.

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Zahrt and Deretic [24] mapped transcriptional and translational start sites. Promoter activity in M. smegmatis culture was considerably lower than that associated with the hsp60 promoter [21]. In M. tuberculosis, mtrA promoter activity was constitutive and did not change on infection of macrophages [24]. However, activity in BCG was far lower than that in M. tuberculosis immediately after infection, but did appear to be induced after infection, since activity after 3 days was far higher than that in M. tuberculosis and comparable to hsp60 promoter activity [24]. Via et al. [19] confirmed induction of the promoter in BCG on infection of macrophages. 2.1.2. Translation Translation initiation is influenced by the structure of the 5 end of the mRNA, the ribosome binding site and initiation codon and the sequences surrounding the latter pair. In general, mycobacterial Shine-Dalgarno sequences are similar in base composition and position to the prokaryote consensus [26]. GUG is used more often as a start codon in mycobacteria than in E. coli, in addition to AUG [14]. For the most part, however, uncertainties around mycobacterial translation initiation have been avoided in the available shuttle vectors by cloning foreign genes as fusions with the 5 end of a mycobacterial gene, including the ribosome-binding site and the initiating codon plus a few of the following codons (e.g. [5]). The use of unmatched promoters and variation in the spacing between promoter and ribosome-binding site may, however, alter mRNA structure. The rate of translation of foreign genes may be influenced by their codon usage. Mycobacterial genomes have a high G + C content: 65.9% in M. tuberculosis, for example [14]. As a result, there is a high degree of bias for codons with G and C at the third nucleotide position. A requirement for codons that are rare in mycobacteria could compromise translational efficiency of foreign genes. Burlein et al. [26] point out, however, that synthesis of Borrelia burgdorferi outer surface protein A (OspA) is still efficient, at 10% of total protein during in vitro culture in BCG [10], despite usage rates for Leucine, for example, of 38% in B. burgdorferi and 1% in mycobacteria. Immunogenicity data confirm that this high level of expression is maintained in vivo. Nevertheless, the optimizing of foreign gene codons to suit mycobacterial expression systems may improve the efficiency of translation and has been used in some studies [27,28]. Abnormal or inefficient post-translational modification and folding will influence the function, solubility and stability of the protein. Accumulation of the foreign protein, whether it is in the correct conformation or not, is determined by a balance between synthesis rate, the modification, folding and export of the protein and degradation by endogenous proteases. 2.1.3. Levels of recombinant protein in rBCG The reported levels of production of recombinant protein in rBCG vary from 15% of total cytoplasmic rBCG protein to approximately 0.1%. Differing expression systems, foreign

antigens, BCG strains and, no doubt, assessment methods account for the range. The studies reporting production levels are reviewed briefly here. Langermann et al. [29] reported 15% (100 ng/106 cfu) levels of pneumococcal PspA in rBCG lysates (hsp60 promoter, cytoplasmic expression, episomal vector). Levels were twoto five-fold lower when the protein was linked to secretion or lipoprotein signals. Stover et al. [5,10] reported a 10% level in lysates of rBCG expressing ␤ galactosidase [5] and Borrelia OspA (20 ng/106 rBCG; hsp60 promoter, cytoplasmic, episomal [10]). Lower levels were detected when OspA was linked to secretion or membrane-anchoring sequences (1–5 ng/106 rBCG [10]). Winter et al. [12] estimated that 1% of cellular protein in recombinant M. smegmatis and BCG was HIV-1 nef (S. albus GroEL promoter, cytoplasmic, episomal). Similarly, Haeseleer et al. [23] estimated that Plasmodium CSP fragment expressed in BCG (BCG 64 kDa antigen promoter, episomal vector) constituted 1% of soluble protein. Mederle et al. [30] expressed SIVmac251 gag from a pBlaF*-driven nefgag operon at 1% levels in episomal vectors and 0.14% levels in equivalent integrating vectors. The episomal vector was, however, unstable. Aldovini and Young [7] estimated that 0.1% of total protein in logarithmic-phase rBCG Pasteur was HIV-1 gag protein (hsp70 promoter, cytoplasmic, episomal). Despite these low levels of protein, intravenous (i.v.) inoculation of mice yielded specific antibody, splenocyte proliferation and CTL responses. Abomoelak et al. [31] reported a 0.5% expression level (5 ng/␮g total protein) with rBCG expressing a pertussis S1-tetanus toxin C hybrid (hsp60 promoter, cytoplasmic, episomal). Expression of the hybrid from the antigen 85A promoter plus signal peptide was 12 times lower. A few studies have attempted to link the different levels of production of the foreign antigen to differing immune responses. In most cases, production level equivalence is based on Western blot band intensity of cell lysates derived from mid-logarithmic-phase rBCG cultures. This does not necessarily reflect the in vivo situation, responsible for induction of the immune response, and such studies need to be interpreted with some caution. However, some do indicate an influence of level of production on, or even a threshold required for, immune response. Abdelhak et al. [32], Lagranderie et al. [33], Abomoelak et al. [31] and Himmelrich et al. [2] compared immunogenicity of similar vectors with various promoters supporting varying levels of production. Abdelhak et al. [32] found that the level of cytoplasmic expression of Leishmania gp63 differed by a factor of 6 when driven by the pBlaF* versus pAN promoters. Despite some differences in the expressed proteins, they suggest that expression level difference is likely to account for protection from L. major lesion growth provided by the pBlaF*, but not the pAN, vector. Similarly, Lagranderie et al. [33] found that serum antibody responses were higher in BALB/c, C57BL6 and C3H mice immunized with rBCG expressing high (pBlaF*-driven), rather than low (pAN-driven),

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levels of ␤ galactosidase. The former rBCG resulted also in higher IFN ␥ production from splenocytes of immunized BALB/c and C3H mice. In their pertussis S1-tetanus toxin C hybrid study, Abomoelak et al. [31] found that expression from the hsp60 promoter was 12 times that from the antigen 85A promoter and that antibody responses were only elicited by the former. Himmelrich et al. [2] compared two different vectors localizing for export (blaF or erp promoter plus signal) expressing different levels of E. coli malE. This is the only study to directly quantify levels and proportions of malE expression in each targeting compartment of BCG (cytoplasm, cell surface, culture medium). Potential drawbacks are that separation of compartments may be compromised during processing and, more importantly, that in vitro quantification of recombinant antigen may not reflect the actual quantity available in vivo. Nevertheless, an earlier and larger antibody response was obtained with the vector expressing and exporting higher levels of malE. Proliferation and IFN ␥ secretion levels also correlated with the level of expression. They noted, in addition, that secreting rBCG strains (blaF-malE signal) produced significantly more malE than non-secreting strains, even when promoters were matched (pBlaF*). They suggest that targeting can influence the level at which heterologous antigens are produced. Mederle et al. [30] expressed SIVmac251 nef-gag from the pBlaF* promoter on both episomal (pAL5000-based) and integrating (Ms6) vectors. Expression from the episomal vector (1% of total protein) was unstable. Expression from integrating vectors (also at 1% with the help of an extra-plasmidic mycobacterial promoter or at 0.14% if isolated from such promoters) was more stable. Interestingly, the isolated integrating vector did not prime a cellular immune response to gag, but the non-isolated integrating and unstable episomal vectors could, suggesting a requirement for a short period of high-level production for such priming. However, only the non-isolated integrating vector produced CD4+ T cell memory responses to gag, indicating persistence as important for this response. The episomal vector produced the lowest levels of anti-gag antibodies. 2.2. Antigen display Another of the key features of the shuttle vectors that would influence immune response to a foreign antigen is the form of antigen display. In rBCG studies, foreign antigen has been expressed cytoplasmically, secreted or linked to the mycobacterial cell membrane. These different forms of display of foreign antigen by BCG cells can influence both the timing of the immune response and the pathway by which antigen is presented to the immune system. Presentation pathways determine the nature of the subsequent cellular and humoral immune responses. Proteins produced within the cytoplasm of BCG remain encapsulated within the bacterium and are not available for processing and presentation to the immune system while the bacterium persists within the macrophage [34]. Immune re-

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sponses against them are, therefore, delayed until the bacterium is killed. At that stage, release of antigen into the phagolysosome will stimulate predominantly CD4+ T cells. While the delay in induction of an immune response may be undesirable for a vaccine, the continuous release of antigen over long periods of time could be useful in sustaining the response [34]. Intracellular processing of antigen may preclude the induction of antibodies. Small peptides (e.g. [35,28]) or naturally-secreted proteins (e.g. [10,29]) have been successfully secreted from rBCG by fusion with secretion signal sequences, usually the mycobacterial ␣ antigen. Secretion of antigen from viable mycobacterial cells into the phagosome results in earlier availability for processing by the immune system and may also limit their degradation by cytoplasmic peptidases. It is expected that antigen processing from the phagosomal space would be via the MHC class II presentation pathway. MHC class II-␣ antigen complexes have, indeed, been demonstrated in macrophage phagosomes within 20 min of mycobacterial infection [36]. The ␣ antigen also, however, stimulates B cell responses (see below). Fusion of foreign antigen to the signal sequence of the M. tuberculosis 19 kDa lipoprotein has been most widely used for directing foreign antigen through and linking it to the BCG membrane. The 19 kDa lipoprotein is highly immunogenic and activates a range of immune responses (reviewed below). Within macrophages, it also trafficks independently of mycobacteria within an hour of phagocytosis [37]. This suggests that an early and varied immune response to foreign antigen linked to this protein is possible. 2.2.1. Secreted antigen Features of the ␣ antigen are briefly summarized below. Native expression is not significant to the study of rBCG vaccines, as foreign promoter elements were commonly used to drive its production. However, the ␣ antigen’s stability, localization and immunogenicity is relevant to a study of foreign antigen linked to an ␣ antigen carrier. ‘Alpha antigen’ has a number of synonyms in the literature: antigen 6 U.S.-J (the U.S.-Japan reference system [38]), the 30 kDa protein, the 30 kDa major secretory (MSP) or extracellular protein [39,40], or, most commonly, antigen 85B [38]. It is expressed from the fbpB gene [41] and its secreted protein number is MPT59 in M. tuberculosis or MPB59 in BCG [38]. The alpha antigen and two highly related proteins, antigen 85A (32 kDa) and antigen 85C (also 32 kDa), make up the antigen 85 complex of secretory proteins. This ‘complex’ reflects genetic relatedness, rather than a physical association between the three genes or proteins. These proteins are expressed by most species of mycobacteria [38] and are the most abundant exported proteins. The ␣ antigen the most abundant of the three, constituting, for example, 22% of extracellular protein in a study by Harth et al. [39]. All three are exported at all stages of culture [38]. There is evidence that expression increases during monocyte infection [41].

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Little is known of their function, although a fundamental role is indicated by their ubiquity. Belisle et al. [42] demonstrated in vitro mycolic acid transferase activity in all three, suggesting involvement in mycobacterial cell wall assembly. All three also have fibronectin binding activity, and associated roles in mycobacterial pathogenesis have been suggested, including promotion of phagocytosis, inhibition of T cell activation or delayed-type hypersensitivity and damage through induction of TNF ␣ (see [38,41]). The ␣ antigen gene is not, however, required for growth of M. tuberculosis [41]. Transcriptional elements of the BCG ␣ antigen gene were identified by Matsuo et al. [43] and Harth et al. [39,44]. The preprotein has 323 amino acids. The first 40 constitute a signal peptide with characteristic amino-terminal basic amino acids followed by hydrophobic domains and an Alanine-X-Alanine signal peptidase recognition site just in front of the mature protein. These features indicate secretion via the general secretory (sec) pathway, which transports unfolded protein. The signal sequence also, however, contains an Arginine–Arginine (RR) motif followed by hydrophobic residues, which suggests a possible alternative of secretion via the twin-Arginine translocation (tat) pathway, which translocates folded proteins [45]. For recent reviews of the mechanisms of protein secretion in Gram positive bacteria, see Tjalsma et al. [45] and van Wely et al. [46]. After secretion, cleavage of the signal sequence releases a 283 amino acid, 30 kDa mature protein from the BCG cell [43]. The ␣ antigen amino acid sequence is highly conserved between mycobacteria [39]. G + C content of BCG ␣ antigen, including the signal, is 64% [43]. The protein is expressed as a monomer, contains one internal disulphide bridge and is not post-translationally modified by addition of carbohydrate or lipid [39]. It is very difficult to degrade by site-specific proteases [39]. Crystal structure has been established [47]. In culture, the ␣ antigen is associated with the mycobacterial cell wall and is released into the culture medium [38]. Antigen 85 complex proteins are also expressed in mycobacteria within monocytes [39]. In M. tuberculosisinfected human monocytes, Harth et al. [39] showed that they localize mainly to the mycobacterial outer cell wall (4.7 particles/bacterium), consistent with a role in cell wall synthesis. They are also found in the cytoplasm of M. tuberculosis (1.6 particles/bacterium), the phagosomal space (0.4 particles/bacterium) and in late endosomes and lysosomes. The alpha antigen is a major target of the immune response to mycobacterial infection. It is, therefore, considered a likely protective antigen for development of an improved tuberculosis vaccine [40,48,49], although Wilkinson et al. [41] warn that it may also have the potential to induce immunopathology. Both B [50] and T cell [51] epitopes have been mapped. MHC class II processing of ␣ antigen begins in phagosomes within 20 min of mycobacterial infection [36]. 2.2.1.1. rBCG fractionation studies: secretion signal. The targeting of foreign proteins linked to secretion signals

within BCG has been analysed in several studies by semiquantitative determination of protein concentration in the cytosolic, membrane, insoluble and culture-medium fractions of rBCG. Stover et al. [10] found that their whole ␣ antigen–OspA fusion was secreted to a limited extent, but was located mainly in the BCG cytoplasm and in a cell wallenriched, insoluble fraction [10]. Very little was located in the membrane fraction. Either the chimaera was largely insoluble or exported protein was trapped in the mycobacterial cell wall. The rBCG surface did stain with antibody. Langermann et al. [29] used the PspA signal sequence to direct export of the PspA protein from rBCG. The product was, however, located primarily in the cytoplasm and was not detected by surface staining with antibody. Himmelrich et al. [2] found, using ELISA, that a proportion of E. coli malE protein linked to export sequences (M. tuberculosis erp, M. fortuitum blaF or E. coli malE export signals) was secreted. The greatest proportion of protein was, however, consistently detected in the cytoplasm. Even so, fluorescence-activated cell sorting (FACS) analysis of antibody-stained cells showed malE at the cell surface of over 90% of bacteria exporting the protein compared to 10% or fewer of cells with membrane-anchoring or cytosolic expression. In all three of these studies, it would appear that export is inefficient, but that it can result in exposure of the foreign protein on the surface of the mycobacterial cell. This export or exposure was sufficient to elicit greater antibody responses [10] or protection [29] than elicited by comparable rBCG expressing the protein cytoplasmically. 2.2.2. Membrane-anchored protein The M. tuberculosis 19 kDa protein (gene Rv3763 [52]) is identical to that of M. bovis and similar to homologues in a range of mycobacteria [52]. The first 22 amino acids constitute a typical lipoprotein signal sequence, indicating secretion via the general secretory pathway (see [45,46]). The protein is lipoylated and glycosylated [53]. Homology with other bacterial lipoproteins indicates that the Cysteine in position 22 is the likely target of acylation, therefore the membrane anchor, and the first amino acid of the mature protein. Lipoproteins are common in mycobacteria, and, given the lack of an outer membrane and the high lipid content of cell walls, may serve to retain protein moieties by anchoring them in the membrane or wall [54]. Young and Garbe [54] postulate that the mycobacterial 19 kDa lipoprotein, in particular, has a role in the transport of nutrients through the cell wall. However, biological function has yet to be demonstrated and sequence comparison has not yielded clear homologies with known proteins [37]. The 19 kDa protein is predominantly associated with the cell wall or cell membrane, rather than cytosolic, fractions of M. tuberculosis cells [53]. During in vitro culture of M. tuberculosis, Young and Garbe [54] have shown progressive accumulation within the culture medium relative to control protein during the logarithmic phase, suggesting a measure of export of the protein from the cells, too. Neyrolles et al.

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[37] noted that the 19 kDa antigen was strongly associated with mycobacterial cells adhering to macrophage-like cells. However, within an hour of phagocytosis of live BCG, M. tuberculosis, recombinant M. smegmatis and recombinant M. vaccae, widespread export of acylated 19 kDa antigen from the phagosome into the host cell occurred. This was dependent on acylation of the protein and on viability of the mycobacteria. In general, bacterial lipoproteins are highly immunogenic [55], which is likely to be due, in large part, to the aminoterminal lipid moiety. Synthetic acylation of peptides and proteins can enhance B, CD4+ T and CD8+ T cell responses [56]. Lipoproteins also stimulate innate responses [57]. The 19 kDa lipoprotein is amongst the most immunogenic of antigens recognized by tuberculosis-infected humans [10]. B and T cell epitopes have been described and 19 kDa-specific B and T cell responses are activated in humans and mice (see [53,58,59,37]). MHC class I presentation of CTL epitopetagged 19 kDa lipoprotein expressed in M. vaccae occurred via a TAP (transporter associated with antigen processing)independent mechanism and also did not appear to involve exocytosis from the infected cell [37]. It did, however, require acylation. The 19 kDa antigen has also been reported to interfere with late MHC class II molecule expression and antigen processing and to abrogate M. vaccae-associated protection against M. tuberculosis in mice [60]. The 19 kDa lipoprotein is, therefore, rapidly and widely distributed from the phagosome, provided it is acylated. It appears to be presented to all arms of immune system, having more access to MHC class I-restricted pathways than is normally associated with BCG infection. Immunogenicity is associated with the lipid moiety. The use of the aminoterminal portion, which contains the acylation site (Cysteine 22), to direct the distribution of foreign antigen within rBCG, therefore, has the potential advantage of rapid induction of a strong and varied immune response. 2.2.2.1. rBCG fractionation studies: lipoprotein signal. Fractionation studies with rBCG expressing 19 kDa signal-linked foreign antigens have confirmed membraneanchoring. Using Western blotting, Stover et al. [10] found most 19 kDa-OspA protein associated with the membrane fraction and very little in either the cytosolic fraction or the medium. Use of the OspA, rather than the 19 kDa, signal sequence, however, resulted in mostly cytosolic distribution, probably a reflection of inefficient processing of a foreign signal peptide by mycobacterial signal peptidase II [10]. Langermann et al. [29] confirmed, using various targeted PspA fusions, that only the 19 kDa signal-linked protein localized to membrane. Nineteen kiloDalton-linked foreign antigen could be detected on the surface of rBCG by antibody staining in both studies and by immunoelectronmicroscopy in the case of OspA [10]. Using antibody staining, Bastos et al. [61] also detected recombinant proteins at the surface of rBCG expressing 19 kDa-linked porcine RRS virus (PRRSV) G5 and M proteins, but not at the surface of rBCG expressing

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the G5 protein cytoplasmically. Himmelrich et al. [2], on the other hand, although confirming expected localization with various signal sequences in an ELISA assay, reported a primarily cytoplasmic location of chimaeric proteins, irrespective of targeting. The 19 kDa-linked malE fusion was detected at the cell surface by antibody staining in only 10% of cells, compared to 90% of those secreting the protein. 2.2.3. The influence of targeting on immune response: comparative rBCG studies Stover et al. [10] compared rBCG expressing OspA in the cytoplasm with those in which OspA was linked to export (␣ antigen export signal and protein) or membrane-anchoring (19 kDa or OspA lipoprotein signal) sequences. The cytoplasmic vector produced most protein in cell lysates. However, specific serum IgG was absent in mice vaccinated intraperitoneally (i.p.) with cytoplasmically-expressed OspA, intermediate in those with cell wall-associated OspA and highest in those with membrane-associated OspA. After boosting, responses were detected in all groups, but were 100–1000fold higher in groups immunized with membrane-associated OspA. These mice were also better protected from challenge with B. burgdorferi than those vaccinated with rBCG expressing either secreted or cytosolic OspA. Langermann et al. [29] compared rBCG expressing cytoplasmic, secreted (PspA signal) or membrane-anchored (19 kDa) PspA. In vitro production was highest for cytosolic PspA and two- to five-fold lower in the others. Regardless of targeting, i.p. immunization of mice led to strong serum IgG responses. However, only the secreting or membraneanchoring rBCG-PspA protected mice against pneumococcal challenge. Hayward et al. [62] compared rBCG expressing the B subunit of E. coli heat-labile enterotoxin (LT-Bh) in either cytoplasmic (hsp60), cell wall-associated (19 kDa promoter and signal) or secreted (hsp60 and ␣ antigen signal) forms. Protein concentrations in cell lysates were similar in all three. After oral immunization of mice (2 × 107 cfu × 3), serum IgA was detected in 19 kDa and secreting groups only. Stool IgA levels were highest with secreted, lower with 19 kDa-linked and lowest with cytoplasmic LT-Bh. Bastos et al. [61] compared rBCG expressing the G5 and M proteins of PRRSV either cytoplasmically or linked to the 19 kDa lipoprotein. After i.p. vaccination of mice (108 cfu), serum antibody and splenocyte cytokine responses were similar for both localization strategies, but neutralizing antibody was elicited only by 19 kDa-linked proteins. Grode et al. [63] compared three rBCG vectors localizing the p60 antigen of L. monocytogenes to the cytoplasm, membrane (19 kDa anchor) or external medium (␣ antigen signal). Expression in each compartment was not analysed quantitatively, but consistency in hsp60 promoter and pAL5000 replicon allowed reasonable assumption of equivalence in protein production potential. Mice vaccinated i.v. with these rBCG were challenged with Listeria. Protection induced by rBCG expressing membrane-anchored (100%) or exported

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p60 (80%) was superior to that provided by cytoplasmicallyexpressed p60 (20%). Listerial load in spleens was lowest in the membrane-anchored p60 group. In vivo depletion of T cell subpopulations at the time of challenge demonstrated that both CD8+ and CD4+ T cells were involved in protection elicited by membrane-anchored p60, while CD4+ T cells only mediated protection by p60-secreting BCG. Consistent with this, ex vivo CD8+ T cell frequencies were highest in groups vaccinated with membrane-anchored p60 rBCG. This indicates that membrane display is important for eliciting CD8+ T cell responses. These studies generally show higher antibody or cellular immune responses elicited by the linking of foreign antigen to targeting sequences rather than expressing it cytoplasmically in BCG. The first two studies described here showed greater responses to targeted antigen despite detection of lower levels of protein in BCG cytoplasmic lysates.

3. Stability of the rBCG vaccine Expression of foreign genes in BCG is not always possible. Lack of success in this respect may be due to overexpression lethality or other forms of protein toxicity. Stover et al. [5], for example were unable to express HIV-1 gp120 (hsp60 promoter) from an episomal vector (n = 5 per cell), but could express the product from an integrating vector (n = 1 per cell). Expression from multicopy plasmid vectors was possible if weaker promoters were used [26]. Langermann et al. [29] were unable to obtain expression of the full-length PspA protein with its natural carboxy-terminal anchor domain (hsp60 promoter, episomal), suggesting that expression of the repetitive domain was deleterious to BCG. The amino-terminal portion was successfully expressed, cytoplasmically, at up to 15% of total protein. Bastos et al. [61] could only detect expression of PRRSV G5 protein (hsp60 promoter; episomal), when 30 amino-terminal hydrophobic amino acids were removed. Kong and Kunimoto [64] could express IL 2 (hsp60 promoter) in rBCG only from integrating, not episomal, vectors. Expression of heterologous antigen within recombinant BCG necessarily imposes a metabolic burden. A portion of the host bacterium’s energy and materials are required to maintain the foreign DNA, express it as RNA or protein and deal with, perhaps destroy, the end product, particularly if it is expressed at a high level or if it is not inert. The extent of this burden will determine the degree to which fitness of the recombinant BCG is compromised. In turn, the relative level of fitness of the recombinant and any derived mutants will determine the rate at which the inserted element (structural instability) or its expression (functional instability) is lost from the bacterial population. Attenuation of the ability of rBCG to replicate and persist relative to wild-type BCG has been reported in vitro and in vivo. Zhu et al. [65] report that rBCG expressing a measles virus nucleoprotein from a single-copy, integrating vector

grew at same rate as wild-type BCG, while those containing a similar multicopy vector (n = 5) had a markedly reduced replication rate. Hess et al. [66] noted that rBCG strains expressing L. monocytogenes listeriolysin persisted within macrophagelike cells for shorter periods than the parental BCG strain. Stover et al. [10] reported attenuation in the ability of rBCG expressing a 19 kDa-OspA fusion to replicate and persist in vivo in mice compared to non-recombinant BCG. Most of the later studies with rBCG include some assessment of stability in vitro or in vivo. This is often provided by an estimate of the percentage of BCG colonies retaining kanamycin resistance, which is used as an indicator of retention of the plasmid. This approach has limitations: retention of selection markers does not, of course, distinguish between retention of intact vector or retention of deletion mutants which have lost the foreign gene but retained the selection marker. This leads to uncertainty when comparing expression levels. More rarely, recovered DNA is mapped or sequenced or, most usefully, levels of production of foreign protein are assessed over time, providing an estimate of functional stability. 3.1. Episomal vectors: in vitro stability Some studies using episomal vectors in rBCG report high levels of stability in vitro. For example, plasmids responsible for expressing M. tuberculosis ␣ antigen in BCG were stable over six consecutive 4-week cultures in the absence of selection and level of expression was unaltered [40]. An ␣ antigen-HIV-1 V3J1 chimaeric protein was secreted by rBCG Tokyo for at least 450 passages in vitro, presumably in the presence of selection [67]. However, in vitro instability of episomal vectors has also been reported regularly. Lim et al. [68] maintained rBCG expressing SIVmac251 env (amino acids, aa, 1–245) stably in culture (pBlaF* promoter), but rBCG harbouring the same vector containing aa 1–521 or aa 215–521 were unstable. In M. smegmatis and M. vaccae, Medeiros et al. [69] analysed ␤ galactosidase activity in a series of episomal promoter probe vectors. 90% of clones carrying an hsp60 promoter-driven expression system lost expression within two subcultures in medium containing antibiotic. The instability rate was lower in M. vaccae, although still considerable at 30% loss within three subcultures. Lower inactivation rates were observed in vectors containing the pAN promoter. Genetic mapping showed gross structural modification in most of these and other vectors, usually in the form of deletions. Not unexpectedly, ␤ galactosidase inactivation rates exceeded the rate at which gross genetic modification was detected indicating that, while mapping is useful for demonstration of changes, it is insufficient to prove integrity. Chawla and Das Gupta [70] reported the disruption of pAL5000-derived vectors in M. smegmatis through transposition of an insertion sequence upstream of the kanamycin resistance gene. This led to structural instability and large deletions. Bastos et al. [61] noted a deletion and frameshift just before the stop codon in one

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isolate of rBCG expressing PRRSV GP5 which resulted in expression of a larger protein. Al-Zarouni and Dale [71] have provided evidence that transient induction of the hsp60 promoter during electroporation may account for the instability associated with the use of this promoter in episomal vectors.

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trials in animals [30]. Once an apparently stable recombinant has been obtained, careful monitoring of stability is required. During manufacture of rBCG-OspA (episomal) for Phase I clinical trials [77], all bacterial samples were analyzed by restriction fragment patterns and the OspA portion of the vector was sequenced at each manufacturing step.

3.2. Episomal vectors: in vivo stability 3.3. Episomal versus integrating vectors Stability of rBCG after in vivo growth has been demonstrated by in vitro analysis of rBCG colonies recovered from immunized animals. Stover et al. [5] recovered BCG-␤ galactosidase (hsp60 promoter) colonies from mouse spleens 2–4 weeks after immunization at more than 30 times the count estimated from initial inoculum. All were resistant to kanamycin and expressed ␤ galactosidase. Murray et al. [72] found that kanamycin resistance was retained in 45% of BCG colonies derived from mice 2 months after rBCG-␤ galactosidase (pAN promoter) inoculation, but only 26% expressed ␤ galactosidase. Retention of kanamycin resistance is, therefore, not necessarily a good indicator of retention of antigen production. In guinea pigs, ␤ galactosidase was produced (pAN promoter) in 34–59% of colonies recovered after 4 months [73]. In studies using foreign antigen other than ␤ galactosidase, Fennelly et al. [74] showed rBCG expression (hsp60 promoter) of a measles nucleoprotein fusion in colonies recovered after 8 weeks in mice. Choi et al. [75] showed expression (hsp60) of EMC-D VP1 in colonies recovered after 8 weeks in mice. Jabbar et al. [76] also demonstrated expression (hsp60) of HPV L1 in three randomly-selected colonies recovered from mice after 8 weeks. Connell et al. [27] confirmed expression (hsp60) of Leishmania gp63 by Western blot in five of five colonies recovered after 10 weeks in mice. Edelman et al. [77] found that all kanamycin-resistant colonies recovered from mice after 9 weeks expressed OspA (hsp60). Furthermore, mycobacteria recovered after 2, 8 and 12 weeks from mice had 53, 17–33 and 8–18% retention of kanamycin resistance. In the human trial with the same recombinant, mycobacteria recovered from swab samples of injection lesions after 14–28 days were 67–80% kanamycin resistant. Finally, Zhu et al. [65] demonstrated measles nucleoprotein in over 90% of kanamycin-resistant colonies recovered from rhesus monkeys months after immunization. Nascimento et al. [78] (pertussis S1 toxin fusion, pBlaF* promoter) did not demonstrate expression after recovery of BCG from mice. However, retention of kanamycin resistance was 86% and 85% at 1 and 2 months after i.p. immunization. Total bacterial numbers recovered from rBCGand BCG-vaccinated mice were comparable, showing that growth and persistence of the recombinant was not compromised. Streit et al. [79], on the other hand, found that only 3% of rBCG expressing Leishmania LCR1 (hsp60 promoter) retained kanamycin resistance 8 weeks after i.p. immunization of mice. It has been suggested that overestimated stability could account for the lack of immune response in some of the reported

It is now well established that integration of vectors into the mycobacterial chromosome tends to result in more stable rBCG. It is likely that both the reduction in expression that accompanies the drop in copy number from five (most episomal vectors) to one (most integrating vectors) as well as better retention, and apparently infrequent mutation, of DNA that is integrated into the genome, contribute to this. Haeseleer et al. [23] compared the maintenance of DNA and expression (hsp60 promoter) of Plasmodium circumsporozooite protein (CSP) from episomal and integrating vectors in M. smegmatis and BCG. Episomal vectors expressing CSP were recovered at low efficiency after electroporation and shown to be structurally unstable. The level of expression from the integrating vector, on the other hand, was consistent over 44 days of pellicle culture in the absence of antibiotic and estimated at approximately 1% of total soluble protein. Kanamycin resistance was maintained for at least 400 generations in M. smegmatis and 50 generations in BCG grown in the absence of antibiotic. The basis on which generation number was estimated from pellicle culture is not clear and details of culture conditions, particularly time spent in stationery phase, are required for a more nuanced interpretation of these results. However, the relative stability of the integrated vector is clear. Interestingly, a second integrating vector encoding a smaller attP and int region was highly unstable, and kanamycin resistance was lost within 2 weeks. Kumar et al. [80] reported a ␤ galactosidase inactivation rate of 2 × 10−3 for MedImmune’s episomal (pMV261, hsp60 promoter) vector containing the ␤ galactosidase gene and a lower rate (1.7 × 10−5 ) for the equivalent integrating (pMV361) vector in M. smegmatis culture. Genetic mapping showed a variety of insertions and deletions. Comparable inactivation frequencies were noted in M. aurum and M. phlei. Mederle et al. [30] compared the structural and functional stability of a synthetic operon containing SIVmac251 nef and gag genes (pBlaF* promoter) in episomal and integrating vectors in BCG. After 60 days in vitro, 55% of colonies transformed with the episomal vector had lost their resistance to kanamycin, but resistance was retained in cultures with integrating vectors after 100 days in culture. It is unclear whether these cultures were passaged and, in the absence of direct measurement of the number of generations and information on growth state, the significance of the number of days in culture is uncertain. However, the relative stability of the integrating vectors is, again, clear. In vivo, kanamycin resistance was retained in 60% of colonies transformed with the episomal vector after 28 days, and 25% after 100 days.

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The comparable levels for two integrating vectors were 85% and 98% after 100 days. Functional stability was measured in colonies recovered from spleen, liver and lungs of i.v.inoculated mice [30]. After 70 days in vivo, 25% of colonies recovered in the episomal experiment still produced protein, compared to 100% of colonies in the integrating group. Loss of expression was correlated with loss of DNA from rBCG transformed with episomal vectors by PCR analysis. Differential maintenance of expression was confirmed by immunohistochemical staining 77 days after inoculation. In contrast to the above studies, however, Springer et al. [81] report high-frequency loss of pMV361-based integrating vectors from M. smegmatis and BCG. During growth of colonies on selection-free plates, approximately 3% lost the plasmid. This occurred in both recA+ and recA− M. smegmatis, indicating that plasmid is lost by a recA-independent mechanism. The loss is a function of the integrase and was prevented when the integrase gene was provided on a second plasmid, separate to that carrying the attachment site (attB) and inserted gene, that was unable to replicate in mycobacteria. 3.4. Stability of expressed protein Evidence for differences in stability of foreign proteins is provided in rBCG co-transcribing SIVmac251 nef and gag [30]. Despite transcription of both in a single mRNA, with nef upstream of gag, nef proteins are detected at considerably lower levels than gag proteins. It was suggested that the gag protein was more stable than the nef protein. Also, many rBCG studies note apparent degradation products of foreign proteins in Western blots (e.g. [23,31,74,78,82–85]). On the other hand, retention of the function of the foreign protein was demonstrated by Kremer et al. [82,86]. Schistosoma glutathione S-transferase (GST) expressed cytoplasmically (hsp60 promoter) in BCG retained its enzymatic activity, strongly suggesting that it had folded properly and retained solubility. A number of studies in which cytokines were expressed in rBCG have also demonstrated cytokine activity in bioassays [64,87–92].

4. Preclinical testing considerations Variables to be considered in the preclinical testing of the rBCG candidate vaccines include dose, immunization route, the strain of BCG used and the animal model used, including potential prior exposure of animals to either BCG or, perhaps, the organism from which the inserted genetic elements are derived. 4.1. Dose Dose–response studies in the rBCG literature are few and all describe antibody responses only. Langermann et al. [93] compared serum antibody titres after intranasal (i.n.) immu-

nization with varying doses of rBCG expressing OspA linked to the 19 kDa antigen. The lowest dose (103 cfu) did not yield antibodies, 105 cfu yielded an intermediate-level plateau in antibody titre and 106 and 108 yielded a high-level plateau, maintained for over 40 weeks. Hayward et al. [62] found that mice immunized i.p. with 106 cfu of rBCG expressing E. coli heat-labile enterotoxin linked to 19 kDa signal sequence showed negligible serum antibody responses, although IgG was induced after boosting. Doses of 107 and 108 cfu led to serum IgG and IgA responses. These two studies do appear to demonstrate a requirement for a minimum, or threshold, dose for such responses. Kremer et al. [86], on the other hand, showed no clear gradation in antiGST antibody responses after i.p. immunization of mice with 5 × 105 , 5 × 106 or 108 cfu of rBCG expressing S. haematobium GST cytoplasmically. Low doses of non-recombinant BCG have been reported to elicit an almost exclusively Th1-type response in mice and higher doses a Th2 response, irrespective of immunization route [94]. Low doses have protected deer from virulent M. bovis where high doses have not [95]. The tendency toward a stronger Th2 response with higher BCG dose may explain the dose-dependency of antibody responses noted by Langermann et al. [93] and Hayward et al. [62]. While large amounts of foreign antigen delivered by high doses of BCG may be desirable for vaccination, the immune responses generated by such doses may be dissimilar to those following vaccination with lower doses. 4.2. Immunization route 4.2.1. In vivo establishment of rBCG after different routes of immunization The manner and extent to which rBCG establishes itself, grows and persists in vivo will determine the magnitude, type, persistence and location (systemic or mucosal) of immune response to the foreign antigen. In vivo establishment depends fundamentally on immunization route. rBCG establishment after administration to mice by various routes has been compared by relative rates of recovery of rBCG colonies 24 h after immunization. Not unexpectedly, recovery after intradermal (i.d.) and i.n. administration is far higher than recovery after oral immunization [73,96,97]. For example, 105 cfu of a 106 cfu i.d. dose of rBCG expressing ␤ galactosidase was recovered at the injection site, while only 200 cfu of a 6 × 106 cfu oral dose was recovered in colon and mesenteric lymph nodes (MLN) [73,96]. In a separate study, Lagranderie et al. [97] recovered 5 × 106 cfu from lungs after i.n. immunization with 3 × 107 rBCG. After oral immunization, viable rBCG are shed in faeces in large numbers for prolonged periods. It appears that rBCG is able to traverse the length of the gut and resist, at least partly, gastric acidity and enzymatic lysis, and that only a small number are taken up by intestinal cells. rBCG-SIV nef administered orally to mice (5 × 109 cfu over 5 days) was shed for 8 days, at over 106 cfu/g on days 3 and 5 [98]. More than 20% of the inoculum of rBCG expressing E. coli heat-labile toxin

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(2 × 107 cfu × 3) was excreted in the 6 h following oral immunization of mice [62]. rBCG-OspA was detected in rectal smears from white-tailed deer 72 h after oral immunization [99]. The spreading and persistence of rBCG administered by various routes has been described by Lagranderie et al. [73,98]. rBCG expressing ␤ galactosidase and administered to guinea pigs by respiratory, oral and i.d. routes established rapidly in the relevant draining lymph nodes and persisted for 16 weeks. By 2 weeks post-immunization (p.i.), rBCG appeared in spleens (all routes) and in lungs (oral or respiratory immunization). rBCG did not persist at these sites beyond 8 weeks p.i. Oral administration of mice with rBCG expressing SIV nef (109 cfu × 5) resulted in early colonization of the pharyngeal lymph nodes and Peyer’s Patches (PP), followed by colonization of the MLN. Except in the PP, rBCG persisted at these sites until 2 months p.i. rBCG appeared in liver, spleen and lungs after approximately 10 days, persisting until 1 month p.i. in liver and lungs, and for over 2 months p.i. in spleen [98]. Administration of BCG intragastrically prevents colonization of pharyngeal lymph nodes and lungs [100]. In mice, Langermann et al. [93] (rBCG-OspA, hsp60) recovered BCG from lungs and spleen 9 weeks after i.n. immunization, but from spleen only after i.p. immunization. Kremer et al. [82] (rBCG-Sm28GST) noted equivalence in numbers recovered in spleen and liver after i.p. or i.n. (5 × 106 ) immunization of mice, but lower recovery from lungs after i.p. immunization. For all routes, most bacteria were eliminated from these sites by 16 weeks p.i. [82]. The in vivo stability data from rBCG studies reviewed in the previous section do not specifically compare the influence of immunization route, but do provide evidence for persistence of rBCG in guinea pigs for up to 4 months [73] and in mice for over 2 months [77]. A few studies have indicated that live rBCG provides better responses or protection than killed rBCG or wild-type BCG mixed with purified antigen, supporting the notion that growth and persistence are important for immune responses. Stover et al. [5] found negligible serum antibody response to heat-killed rBCG containing tetanus toxin fragment C, while the live rBCG produced high titres that increased over the 12week study period. Heat-killed rBCG did not protect against challenge with tetanus toxin, while live rBCG partially or completely protected [101]. Horwitz et al. [40] reported that immunization with BCG overexpressing ␣ antigen provided superior protection of guinea pigs than wild-type BCG mixed with purified protein. Streit et al. [79] reported better protection of mice from Leishmania challenge with BCG expressing LCR1 than with LCR1 protein alone. Choi et al. [75] reported lower shortterm immune responses, but superior long-term responses with rBCG expressing EMC-D virus VP1, compared to a subunit VP1 vaccine. On the other hand, Jabbar et al. [76] got better responses with HPV protein plus adjuvant than with rBCG-HPV, and suggested that rBCG may be useful in

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priming or in retention of memory responses, but that it is not useful as a single-component vaccine. 4.2.2. Immune response and immunization route Many of the rBCG studies have attempted to compare responses to foreign antigen expressed by rBCG after administration of the same rBCG via different routes. Clear comparisons are difficult, though, as doses used for each route are usually different. Kremer et al. [82] did, however, administer rBCG-Sm28GST to mice at the same dose (8 × 106 cfu) by i.n., i.v., i.p. and subcutaneous (s.c.) routes. Stronger serum antibody responses were elicited by i.p. and i.v. routes and IgG, but not IgA, isotype distribution varied with route. Even at equivalent anti-Sm28GST antibody levels, i.p.-immune serum (mixed IgG isotypes) had stronger neutralizing activity than i.v.- or i.n.-immune sera (predominantly IgG2a or Th1-type), which may be related to these isotype differences. The i.n. route triggered the highest anti-BCG response, consistent with a Th1-type response. Antibody responses following s.c. immunization developed far more slowly than those following immunization by the other routes. Responses persisted for more than a year. Other studies specifically comparing routes of administration at different doses are listed only briefly here. Lagranderie et al. [73] compared oral (6 × 1010 cfu × 3), respiratory and i.d. (106 ) routes of administration of rBCG-␤ galactosidase in guinea pigs. DTH responses to ␤ galactosidase and lymph node cell proliferation were higher after oral and respiratory immunization. Hiroi et al. [102] compared serum antibody after nasal (3 × 10 ␮g), oral (3 × 100 ␮g) and s.c. (3 × 100 ␮g) immunization with rBCG Tokyo-HIV-1. Peak titres were comparable, but antibody persisted for longer after i.n. and s.c., rather than oral, immunization. Kawahara et al. [67] compared a complex set of routes (i.d., i.n., intrarectal (i.r.), i.r./s.c., i.r./i.d.) to immunize guinea pigs with rBCG-HIV-1-V3J1 and obtained superior cellular and humoral responses with route combinations. In a comparison between parenteral routes only, Abdelhak et al. [32] noted equivalent protection of rBCG-gp63 against L. major lesion growth whether administered s.c. or i.v. to mice. The studies of Langermann et al. [93], Lim et al. [68] and Lagranderie et al. [97] compared induction of mucosal immune responses after immunization by various routes. Briefly, Langermann et al. [93] compared i.n. (108 cfu × 2), intragastric (107 cfu) and i.p. (108 cfu) immunization of mice with rBCG-19 kDa-OspA. I.n. and intragastric immunization led to IgA responses in lungs, intestinal lamina propria (LP) and vagina. These were strongest after i.n. immunization. The intragastric route yielded IgA in faeces. No mucosal responses were detected in lungs or vagina following i.p. immunization. Lim et al. [68] compared oral (6 × 1010 cfu) and s.c. (109 cfu) immunization of guinea pigs with BCG-SIV env. Higher levels of faecal IgA were obtained after oral immunization. Lagranderie et al. [97] compared oral (1.5 × 109 cfu × 3), aerogenic, nasal (107 cfu × 3) and rectal (3 × 108 cfu × 3) immunization of

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mice with rBCG-SIVmac251 nef/gag/env. The rectal route produced the highest intestinal IgA responses. In order to elicit an intestinal mucosal immune response, a mucosal immunization route is indicated. Oral or intragastric immunization is inefficient, in that much of the dose is lost, but it does lead to efficient intestinal response. In guinea pigs, the PP and MLN are rapidly colonized by rBCG after oral immunization. rBCG does not persist for long in PP, but does appear to persist for 2 months in MLN [98]. Since this is a mucosal inductive site, the continued stimulation of a response at mucosal effector sites is possible. Continued stimulation of systemic responses is theoretically possible for a number of months. 4.3. BCG strain and preparation There are a number of strains of BCG (e.g. Pasteur, Tokyo, Danish, Glaxo, Connaught, Moreau) in use for vaccination against tuberculosis [103], a legacy of widespread distribution and use of BCG vaccine prior to the establishment of seed-banking systems. The genealogy of the strains has been traced by Oettinger et al. [103] and Behr and Small [104]. There has been some consideration of whether the extent of expression of foreign protein and the strength of immune response depends on the strain of BCG used. For example, Burlein et al. [26] noted that the expression of foreign genes from the same vector varied widely within different strains of BCG. OspA expression from pMV261, for example, could be detected in some strains only. The same gene in a different vector was detected in a different combination of strains. The authors suggested that some strains, such as BCG Tokyo, expressed or accumulated protein more poorly than others, such as BCG Pasteur. Hanson et al. [105] also reported more reliable coexpression of PspA and PAL in BCG Pasteur than in BCG Connaught. Al-Zarouni and Dale [71] noted that BCG Moreau appeared to be more susceptible to deletions than the Pasteur or Tokyo strains. However, in the absence of supporting data on plasmid stability in, and growth and identity of, the strains under comparison, much of this evidence should be treated with caution. The existence of significant differences in gene expression between BCG strains remains uncertain. Lagranderie et al. [106] compared five different strains of non-recombinant BCG (Pasteur, Glaxo, Tokyo, Russian, Prague) in mice and demonstrated differences in immunogenicity between them, suggesting that choice of BCG strain may influence immunogenicity independently of expression capability. For example, in mice, the Tokyo and Prague strains appeared to generate weaker cellular responses, were eliminated more rapidly and were less effective in eliminating a mycobacterial challenge (rBCG expressing ␤ galactosidase, in this case) than the Pasteur strain. Behr [107], however, cautions that there is insufficient evidence at this stage to easily suggest differences between BCG strains in either immunogenicity or adverse effects. AlZarouni and Dale [71] found no marked difference in expres-

sion of foreign antigen in three BCG strains where plasmids were shown to be intact. Differing methods of rBCG preparation in different laboratories may also influence immunogenicity [108]. The growth stage of the bacteria at harvest, the extent of bacterial clumping, the ratio of live to dead cells and the amount of soluble antigen may be significant. 4.4. Animal model As a result of differing susceptibilities to BCG infection, controlled by the bcg gene in mice, as well as other genetic influences, the strain of mouse in which rBCG is tested is significant. A number of rBCG studies have demonstrated this. Stover et al. [10] screened for responsiveness to BCG-OspA in 24 strains of inbred and outbred mice and found that sensitive (s) and resistant (r) allelles to BCG growth did not correspond with presence or absence of antibody response. BALB/c (s), C3H (r) and Swiss Webster (r) mice demonstrated intermediate, high and low responses, respectively, to BCG expressing OspA cytoplasmically. BCG expressing membrane-associated OspA elicited higher and more uniform responses. Consistent with this, Langermann et al. [29], using similar vectors, reported comparable antibody responses and protection in C3H and BALB/c mice immunized with BCG recombinants secreting or membrane-linking PspA. Lagranderie et al. [33] reported strong antibody responses to lower levels of ␤ galactosidase expression in BALB/c mice rather than C57BL6, C3H and CBA mice. Splenocyte IFN ␥ production was highest in C3H and lowest in C57BL6 mice and recovery of BCG from spleens lowest in C57BL6 mice. Matsumoto et al. [83] reported that immunization with rBCG expressing the C-terminal fragment of MSP-1 protected C3H/He and A/J mice, but not C57BL6 mice, from P. yoelii challenge. Janssen et al. [109] noted that BALB/c mice produced lower levels of IFN ␥ than C57BL6 mice after vaccination with rBCG expressing an immunodominant peptide from house dust mite. Guinea pigs are far more permissive hosts for BCG growth than mice. Guinea pigs, like humans, but unlike mice, are susceptible to low aerosol doses of M. tuberculosis and develop disease that closely resembles tuberculosis in humans clinically, immunologically and pathologically [40]. They are, however, far more susceptible to disease after infection than humans and are used, therefore, as a sensitive model in studies assessing protection against tuberculosis. 4.5. Prior exposure of animals to BCG or foreign antigen Since BCG vaccination has been widely used, an important issue in development of rBCG vaccines is whether prior immune responses to BCG or to environmental mycobacteria would interfere with responses to subsequent rBCG immunization. Prior exposure to large numbers of environmental

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mycobacteria is likely curtail immune responses to BCG vaccine, as has been demonstrated in animals [110,111]. Gheorghiu et al. [112] compared immune responses to rBCG expressing ␤ galactosidase or HIV-1 nef antigens in na¨ıve mice with those of mice previously exposed to BCG. Prior BCG exposure limited growth of rBCG in spleen or lymph nodes and decreased proliferative responses against the foreign antigens, although the latter suppression never exceeded 50%. On the other hand, anti-␤ galactosidase antibody responses were consistently enhanced and antibody isotypic profile was not altered. They concluded that prior immunization with BCG would not limit the use of rBCG vaccines. Kameoka et al. [113] also demonstrated that MHC class I-restricted CTL responses to rBCG expressing V3 env CTL epitope were not diminished by prior experimental infection with non-recombinant BCG. Prior exposure of animals to the foreign antigen cloned into rBCG is also potentially significant, as pre-existing immune response may interfere with the development of an immune response to rBCG expressing the relevant protein. In laboratory experiments, preinfection can be avoided by breeding animals in the appropriate isolation facilities.

[7]

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[9]

[10]

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[12]

[13]

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5. Conclusion A wide range of rBCG vaccines have been constructed and evaluated, primarily in animal models. These studies have enabled this analysis of some of the variables that may influence immunogenicity. We hope that this review will assist in defining and, perhaps, limiting the study-to-study variables in future rBCG experimentation.

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Acknowledgements [19]

The authors gratefully acknowledge the support of the South African National Department of Health, the Medical Research Council and the National Research Foundation.

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