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XIX. A transphyletic anti-infectious control strategy based on the killer phenomenon

Stefania Conti, Walter Magliani, Mara Gerloni, Antonella Salati, Elisabetta Dieci, Simona Arseni, Paola Fisicaro, Luciano Polonelli
DOI: http://dx.doi.org/10.1111/j.1574-695X.1998.tb01200.x 151-161 First published online: 1 September 1998


A strategy for the prevention and control of candidiasis, pneumocystosis, and tuberculosis, based on the idiotypic network of the yeast killer effect has been envisaged. Anti-idiotypic antibodies representing the internal image of a candidacidal, pneumocysticidal, and mycobactericidal killer toxin from Pichia anomala and idiotypes of killer toxin-neutralizing monoclonal antibodies mimicking the specific cell wall receptor of sensitive microorganisms might provide a unique approach for engineering innovative antibiotics and vaccines active against taxonomically unrelated pathogenic microorganisms. The rationale of the strategy relies on a phenomenon of microbial competition which has been mutated by the immune system in the response to natural infections.

  • Yeast killer phenomenon
  • Candidiasis
  • Pneumocystosis
  • Tuberculosis
  • Anti-idiotypic therapy
  • Idiotypic vaccination

1 Introduction

Nowadays, opportunistic infections are the cause of emerging and re-emerging life-threatening diseases, particularly in AIDS patients, but also in HIV-uninfected immunocompromised individuals for which the ordinary prevention and therapeutic measures often fail or are insufficient [1]. In particular, the incidence of tuberculosis has increased in many endemic areas or spread to populations of industrialised countries throughout the world [2]. Within a few decades, more than 90 million cases and 30 million deaths due to tuberculosis are expected [3]. Owing to the difficulty to treat all patients with effective chemotherapy, which might be prolonged for at least 6 months with several antibiotics, an increasing number of multi-drug resistant (MDR) strains of mycobacteria are isolated [4]. On the other hand, different trials of the protective efficacy of the BCG vaccine reported an average efficacy of only 50% and its impact on the tuberculosis trends worldwide remains unclear [5]. Thus new antibiotics and vaccines for the treatment and prevention of tuberculosis are intensely sought for. Candida albicans and Pneumocystis carinii are important opportunistic pathogens, especially in HIV-infected patients. Yeast belonging to the genus Candida are responsible for vaginal infections in one third of fertile women as well as for AIDS-related oral and oesophageal candidiasis. C. albicans also represents an important agent of difficult-to-treat deep-seated infections in neutropenic, bone-marrow transplanted patients [6, 7]. P. carinii pneumonia is a severe pulmonary disease and the major cause of death in AIDS patients [8]. No effective vaccine has been currently validated to protect against candidiasis and pneumocystosis. Available antibiotics against Mycobacterium tuberculosis, C. albicans and P. carinii are frequently associated to secondary untoward effects (pneumocystosis) or loose their efficacy with developing microbial resistance (tuberculosis, candidiasis). In addition, since immunocompromised patients are prone to several opportunistic infections, prophylaxis or therapy often involve the administration of combined anti-microbial drugs targeted to different pathogens thus increasing the likelihood of negative drug interactions. A rationale approach of complementary transdisease vaccination and therapy underlying such different pathogens as M. tuberculosis, C. albicans, and P. carinii would be of great relevance from a theoretical and practical point of view.

2 Rationale

A potential unique approach for the prevention and treatment of multiple opportunistic infections could be based on the idiotypic mimicry of yeast killer toxins with a wide spectrum of anti-microbial activity and their specific receptor occurring in the cell wall of sensitive microorganisms as well. The selected strain of Pichia anomala (ATCC 96603) secretes a killer toxin (KT) endowed with a wide spectrum of microbicidal activity against taxonomically unrelated pathogenic microorganisms such as C. albicans, P. carinii, and M. tuberculosis, including the multi-drug resistant (MDR) strains [9]. Even though KT displayed a marked therapeutic effect when topically applied in animal models of experimental fungal infections [10], it proved to be very labile at physiological temperature and pH [11] and could not be used in vivo as systemic antibiotic because, as expected of large, foreign proteins, it was both toxic and antigenic [12]. This apparent insurmountable limit was then approached by the idiotypic network theory assuming that the steric interaction occurring between KT and specific cell wall receptors (KTR) present on the sensitive microorganisms could be mimicked by idiotypic—anti-idiotypic antibody complementarity [13].

Monoclonal antibodies were produced from mice immunised with KT and one of them (mAb KT4), characterised as an IgG1, proved to be neutralizing the fungicidal activity of KT against a C. albicans reference strain [14]. MAb KT4 has been used indeed to obtain in different animal models anti-idiotypic antibodies (KTIdAb) which were able to mimic the killer activity of KT. In fact, they exerted a microbicidal effect on C. albicans cells and allowed the visualisation of the putative KTR mainly located on the hyphal forms and budding cells of the yeast [1517]. The major advantage of such KTIdAb is that they are able to exert their anti-microbial activity at physiological conditions. Monoclonal (mAb KTIdAb) and recombinant antibodies in the single chain format (ScFv KTIdAb) were also produced by the spleen of rodents immunised with mAb KT4 by the hybridoma and DNA technology respectively [18, 19]. One mAb KTIdAb, termed K10, was active against C. albicans, P. carinii, and M. tuberculosis in vitro [18, 20]. This activity, which was neutralised by mAb KT4, proved to be mediated by binding of mAb K10 to KTR of the different pathogenic microorganisms. MAb K10 was curative in an experimental model of candidal vaginitis and displayed a therapeutic effect when given by aerosol to nude rats experimentally infected by the intratracheal route with P. carinii[19]. A selected ScFv KTIdAb, named H6, was obtained by using the recombinant phage antibody system. ScFv H6 showed the highest killer activity against C. albicans cells in vitro and therapeutic potential in experimental rat candidal vaginitis [19]. ScFv H6 proved to be also active against P. carinii and M. tuberculosis and bound to KTR of the cell wall of susceptible microorganisms [20]. All of the effects of mAb K10 and ScFv H6 were significantly neutralised by mAb KT4 and all of them competed with KT for binding to KTR.

MAb KT4 was also used as a parenteral idiotypic vaccine for the production of KTIdAb in mice which were protected against lethal intravenous inocula of C. albicans cells [21]. Likewise, a significant protection against candidal vaginitis was obtained in female rats intravaginally vaccinated with mAb KT4 [22]. The level of immunoprotection in the two animal models correlated with the serum or vaginal titres of KTIdAb. Secretory KTIdAb from the vaginal fluids have been shown to confer passive immunoprotection to naive animals experimentally infected with C. albicans cells [22]. This is the first evidence that prevention (by idiotypic vaccination) or therapy (by passive KTIdAb administration) proved to be successful by the exploitation of the killer phenomenon, an event occurring in the competition of microorganisms in natural habitats [23]. Nature has probably taught us how to exploit this phenomenon through the idiotypic network for host—parasite relationship [24].

3 The transphyletic receptor

The internal imaging of KT by KTIdAb necessarily implies the imaging of KTR by the idiotype of mAb KT4. Theoretically, in the course of infection with KT-sensitive microorganisms, the immune system should, therefore, be able to recognise the conformation of the specific KTR similarly to the idiotype of the KT-neutralizing mAb KT4. This hypothesis was experimentally confirmed by the induction of serum and secretory anti-KTR, KT-like antibodies (KTAb) by repeated parenteral or mucosal immunisation with KTR-bearing C. albicans cells [25]. Natural KTAb were also detected in the antibody repertoire of individuals undergoing recurrent candidal infections, and in the vaginal fluid of women affected by recurrent vulvovaginal candidiasis [25]. These KTAb proved to be candidacidal in vitro and to confer passive immunoprotection to rats experimentally infected with C. albicans cells [25]. They have also proven to inhibit the P. carinii in vitro attachment and in vivo infectivity [26, 27], and to kill, in vitro, a MDR isolate of M. tuberculosis[20].

These events suggest a functional homology of human natural KTAb in the three different microbial systems. Human natural KTAb proved to compete with KT for binding to KTR on C. albicans cells, and particularly in the hyphal form and budding cells as well as on P. carinii and M. tuberculosis cell wall [20, 27]. The functional identity between KTAb induced by immunisation with KTR-bearing microbial cells and KTIdAb induced by mAb KT4 vaccination has been determined by the ability of C. albicans cells to boost anti-idiotypic response in animals immunised by idiotypic vaccination and by the immune purification of microbicidal KTAb on mAb KT4 [25]. The microbicidal activity of human natural KTAb was significantly neutralised by adsorption with mAb KT4. The structural homology between KTR of C. albicans and M. tuberculosis has been envisaged by the lack of binding to mycobacterial cells of KT and KT-like antibodies after adsorption with KT-sensitive C. albicans cells [20].

4 Transdisease prevention strategy

A transdisease vaccination strategy based on the occurrence of a transphyletic KTR in such different microorganism as C. albicans, P. carinii, and M. tuberculosis, as well as the KTR-like idiotype of mAb KT4 may be envisaged through different molecular approaches. They basically are the cloning and expression of the common transphyletic KTR in the diverse pathogenic microorganisms and its internal image idiotype of mAb KT4; the demonstration that recombinant KTR or KTR-like idiotype of mAb KT4 are effective vaccines in experimental models; the assessment of immunisation with KTR or idiotype of mAb KT4 encoding genes in animal models.

5 Molecular cloning and characterisation of the idiotype of mAbKT4

The cloning and expression of the genes encoding the idiotype of mAb KT4 might be performed by using a phage-display system. Purified mRNA isolated from mAbKT4-producing hybridoma cells should be reverse-transcribed, and the heavy and light chain antibody genes amplified by PCR. The VH and VL genes could be assembled into a single chain gene, cloned in the phagemid vector pCANTAB 5 E and expressed as fusion proteins on the phage tip. The specific phage-displayed antibodies might be selected and enriched by panning against P. anomala KT and their binding activity to the toxin could be evaluated in an enzyme-linked immunosorbent assay. The selected antibody genes should be sequenced and used in the somatic transgene immunisation. The cloned KT4 single chain genes might be inserted in different mammalian expression plasmids driven by viral promoters (e.g. CMV, RSV) or in immunoglobulin genes under the control of specific promoter and enhancer elements.

Immunofluorescence studies demonstrated that KTR is at the cell surface of C. albicans. The capacity of mAbK10 to kill C. albicans in vitro and in vivo, demonstrated that the KTR is a functional target, especially on the hyphal forms of the fungus. For this reason, a cDNA expression library with mRNAs from C. albicans hyphal forms is an optimal candidate for immunoscreening and cloning the relevant KTR gene by using mAbK10.

Several cDNA expression libraries in λgt11 and λZAP phages from C. albicans hyphae at different times of elongation have been recently obtained [28]. These libraries might be screened against KT-like Ab and positive clones could be subcloned and express in a suitable Escherichia coli expression vector or in a eukaryotic (Pichia pastoris) expression model. Alternative and integrative to this approach might be the extraction and purification of natural KTR form hyphae, starting from α-glucanase-treated cell walls. Immunoaffinity purification with mAbK10 may allow direct sequencing, by Edman procedure, of enzyme-cleaved KTR fragments, and use of PCR degenerated oligonucleotide primers for KTR cloning. Given the potential homology in KT-sensitive microorganisms, the KTR DNA sequences characterised in P. carinii or M. tuberculosis could be made available for constructing PCR primers and suitable probes to amplify and identify the relevant KTR gene in C. albicans (see below).

6 Molecular cloning and characterisation of the transphyletic KTR from Mycobacterium mutant library

The gene encoding KTR in M. tuberculosis might be isolated by screening of a mutant library of M. tuberculosis and other mycobacteria for mutants that are resistant to KT-like Ab. The mutant library would be constructed by random mutagenesis using a transposon based system. Several mycobacterial insertion sequences and transposons have been isolated and properly characterised. One of these elements, IS6100, was successfully used for generating representative insertion libraries in the non-pathogenic fast-growing Mycobacterium smegmatis species [29]. A thermosensitive (ts) mutant of the mycobacterial plasmid pAL5000 was used as a delivery vector in these experiments. Vectors allowing for the selection of large numbers of transposition events in M. tuberculosis have been developed. These vectors take advantage of the counterselectable marker sacB. This gene from Bacillus subtilis encodes a secreted levansucrase, which catalyzes hydrolysis of sucrose and synthesis of levans [30]. In E. coli and other Gram-negative bacteria, the production of SacB in the presence of sucrose is lethal [31]. This sensitivity to sucrose has been used as a powerful way to select the loss of a plasmid carrying sacB[32]. This gene, expressed in M. smegmatis or in M. tuberculosis complex, gives a complete growth inhibition when mycobacteria are cultivated in the presence of sucrose [33]. This system was recently shown to be suitable for randomly inserting a transposon in M. tuberculosis and in BCG. The phoA reporter has been used successfully to isolate gene encoding exported proteins. In case the KTR is a protein, it will be of interest to construct phoA fusions with the receptor gene. The role of the different genes that will be hypothesised by the presence of open reading frames in the genetic region identified by the methodology described above, could be investigated by disrupting them and analysing the resultant phenotype [34].

In C. albicans, KTR has been shown to be a protein, and its characterisation is ongoing (data not reported). Determination of its amino acid sequence will allow to search for similar sequences in M. tuberculosis protein sequences deduced from the M. tuberculosis genome project, now at a very advanced stage. This might enable the investigation of the genetic regions involved in sensitivity to KT-like Ab. In addition and in alternative, when C. albicans KTR genes would be cloned derived DNA probes could be used for attempting the identification of the KTR gene in P. carinii and M. tuberculosis genomic libraries.

7 Molecular cloning and characterisation of the transphyletic KTR from P. carinii

The identification and characterisation of KTR from P. carinii might be carried out by testing mAb K10 against both total lysate and different subcellular fractions, permitting an estimation of the size of the P. carinii KTR, an assessment of the extent of glycosylation and the possible localisation to a particular subcellular fraction, for example the cell membrane fraction.

A number of libraries, both genomic and cDNA, from rat-derived P. carinii (P. carinii sp.f. carinii) in bacteriophage lambda are currently available. These libraries could be screened using the different KT-like Ab, in order to identify the gene encoding the KTR in P. carinii. Positive clones should be selected for further characterisation. As an alternative approach, sequence data obtained from the KTR studies being carried out on Candida and Mycobacterium should be taken into consideration. Probes could be designed for evaluation on Southern blots of restriction endonucleolytic digested genomic P. carinii DNA and also of P. carinii chromosomes separated by pulsed field gel electrophoresis. DNA probes which show positive hybridisation might then be used to screen both genomic and cDNA P. carinii libraries. In another approach, based on DNA homology among the genes encoding the KTR from different pathogens, KTR DNA sequences obtained from Candida or Mycobacterium could be used to design degenerate PCR primers, in order to amplify a fragment of the gene from P. carinii. The PCR product thus generated might be used to screen both the genomic and cDNA P. carinii libraries [3539].

The DNA sequence of the candidate clone should be determined and the derived amino acid sequence established. These sequences might be compared to those in the GenBank and EMBL data bases in order to identify any homologous sequences. Confirmation that the sequence is of P. carinii origin could be obtained by probing P. carinii chromosomes, separated by pulsed field gel electrophoresis, with the cloned gene sequence. In addition, endonuclease restriction digests of P. carinii genomic DNA should also be probed. These data could also give an indication on the copy number of the candidate gene in the P. carinii genome.

The gene should be subcloned into an E. coli expression vector, for high level expression of the candidate sequence. In the event of this being unsuccessful, a yeast or a baculovirus expression system might be used. When high levels of expression should have been achieved, the recombinant protein could be recovered and purified. This would yield high concentrations of purified protein for a variety of biochemical studies, such as binding studies and inhibitor studies. Further large scale expression and purification should be carried out to produce sufficient amounts of recombinant protein to attempt crystallisation, for the determination of the three-dimensional structure of the molecule.

At each stage of the project, the data obtained from the identification of the KTR of P. carinii might be compared to data from the other KT-sensitive pathogens being analysed. The proposed investigations should be carried out initially using P. carinii sp.f. carinii. In the long term, however, it is essential that the KTR of human-derived P. carinii, (P. carinii sp.f. hominis), is identified. It has been demonstrated that high levels of divergence exist between P. carinii sp.f. carinii and P. carinii sp.f. hominis[4043]. However, to date, genomic and cDNA libraries of P. carinii sp.f. hominis have been shown to be of poor quality due to the inability of obtaining sufficient numbers of organism, since P. carinii cannot be cultured in vitro. Nonetheless, the KTR of P. carinii sp.f. hominis is likely to be homologous to that of P. carinii sp.f. carinii since KT-like Ab stain, in immunofluorescence, the organisms present in human bronchoalveolar lavages [27].

8 Intracellular immunisation by recombinant chimeric proteins

Antennapedia (Antp), a transcriptional factor involved in the morphogenesis of Drosophila embryos has been fortuitously shown to translocate across intact cell surface membranes [44, 45]. This property has been mapped to its aminoterminal homeodomain that consists of 60 amino acids. The translocation of Antp into the cytoplasm occurs in wide rage of temperature between 4 and 37°C and does not utilise the endocytic pathway. Synthetic peptides of Antp made of d-amino acids are also able to cross the cell membrane thus ruling out the possibility that Antp is translocated through a receptor-mediated mechanism involving a chiral interaction [46]. The property of Antp to translocate across the cell membrane has been exploited to vehiculate synthetic peptides containing T-cell epitopes of the nucleoprotein of the influenza virus inside antigen-presenting cells (APC). These peptides were shown to induce a potent cytotoxic T-cell response in the immunised mice [47]. Chimeric proteins of more than 350 amino acids containing Antp at their amino terminal end can easily translocate across the cell membrane.

It would be of great interest to exploit the ability of Antp to translocate across the membrane to develop an antigen delivery system that is able to elicit a humoral and cell-mediated (helper and cytotoxic) immune response against the KTR of KT-sensitive microorganisms or the KTR-like Id mAbKT4. The experiments could aim at generating chimeric proteins containing the homeodomain of Antp genetically linked to the sequence of KTR or KTR-like Id mAbKT4. In order to achieve this goal, different approaches might be planned: (1) express recombinant chimeric molecules Antp-KTR or Antp-Id mAbKT4 in E. coli; (2) assess the ability of the chimeric molecules to translocate across the cell membrane of APC.

9 Development of lactic acid bacteria as carrier systems presenting new transphyletic KTR or mAbKT4 idiotype

The development of efficient local vaccines represents a top priority in several vaccination programmes as they usually are associated with fewer side effects and would facilitate vaccination of large populations by reducing the need for trained personnel. The aim is to neutralise the mucosal pathogen at the initial point of infection, i.e. at the mucosal surfaces of external body cavities.

Lactic acid bacteria (LAB) may well represent an ideal carrier for this purpose as these organisms offer some major advantages: (1) dietary LAB are GRAS (Generally Recognised As Safe) organisms: they are widely used in the preparation of food and feed products; (2) their health beneficial role (probiotic effect) has been documented; (3) Lactobacillus is a normal commensal of specific body cavities such as the gut, urinary tract and vagina; (4) Lactobacilli often possess intrinsic adjuvant activity; (5) large scale production does not present major obstacles; (6) production prices are expected to be lower than those for the vaccines currently on the market.

A major objective in this perspective could be the development of LAB strains into safe live vector expressing the KTR gene of KT-sensitive microorganisms or the Id mAbKT4 gene. More specifically, the study could be focused on the construction and immunological evaluation of LAB strains for the production and topical delivery of these antigens in the vagina or the intestine in such a way that both mucosal and systemic specific immune responses are generated. This might be carried out with strains of Lactobacilli colonizing the vagina or the gut which would be selected on the basis of physiological, ecological and genetic criteria.

10 Somatic transgene immunisation by KTR or mAbKT4 idiotype

Somatic transgene immunisation (STI) is a new method of DNA immunisation that targets B-lymphocytes [48, 49]. A single intraspleen inoculation of plasmid DNA carrying a full length Ig heavy (H) chain gene triggers reproducibly: (1) expression of the transgene in the spleen for 3–4 months with uptake and selective persistence of the transgene in the splenic lymphocytes in an integrated form; (2) secretion of the transgene Ig (TgIg) in the serum; and (3) induction of durable immunity against TgIg with persistent immunologic memory [50].

A new strategy in vaccination against Candida, Pneumocystis, and Mycobacterium that results from the combination of two new concepts: idiotypic vaccination and somatic transgene immunisation might be pursued.

STI is a rationale method to initiate an immune response based on the fact that the transgene, an immunoglobulin (Ig) under the control of tissue-specific regulatory elements, once inoculated into a lymphoid organ transduces B-lymphocytes. It has been shown that antibodies against the transgene product only develop when the transgene is inoculated directly into a lymphoid organ (e.g. the spleen) [48, 51]. For this reason, as a model of systemic immunisation, plasmid DNA could be inoculated under the control of Ig promoter and enhancer elements for tissue-specific expression via the intrasplenic route. In comparison, to evaluate how the route of DNA inoculation affects the ability to raise protective titres of anti-idiotypic antibodies, plasmid DNA containing viral promoters (e.g. CMV or RSV) could be inoculated by intramuscular and the intradermic routes. A way by which STI could be modified is by the use of a delivery system, such as polymers, that can compact DNA and enhance cellular uptake. In addition, the plasmid DNA may be targeted to specific tissues, such as mucosal sites for the induction of mucosal and systemic immunity.

For comparative purposes, as a model of mucosal immunisation, the effect on immunogenicity of microspheres of hyaluronic acid as vehicle for intranasal delivery of plasmid DNA under the control of either viral promoters or immunoglobulin (Ig) promoter and enhancer elements might be studied.

In addition, the priming of humoral and cellular responses and immunologic memory against transphyletic KTR and Id mAbKT4 administered to mice as plasmid DNA by different routes of inoculation might be investigated.

11 Evaluation of immunoprotection of KTR and mAbKT4 idiotype based vaccines

The recombinant KTR or Id mAbKT4 products as well as their gene constructs might be used for vaccination to generate candidacidal antibodies and cell-mediated immunity in two well-validated animal models of candidal infection, i.e. the rat vaginal infection, which is an oestrogen-dependent mucosal infection mimicking acute human vaginitis, and a deep-seated mouse infection by Candida in cyclophosphamide-immunodepressed neutropenic animals or neutrophil-depleted mice by the injection of anti-neutrophil mAbs [52], which are both correlates of serious systemic infections in leukaemic, bone marrow-transplanted patients [53, 54]. The capacity of mAbK10, to which the anti-KTR antibodies are logically expected to resemble, of exerting a curative effect in at least one of the above models (the Candida vaginitis) has already been documented [19].

Appropriate positive and negative controls could be inserted using other immunogenic and protective candidal antigens and irrelevant immunogens (polysaccharides from Bacteroides fragilis and mannoproteins from Saccharomyces cerevisiae) [55].

Immunogenicity and protective efficacy of the recombinant KTR or Id mAbKT4 products as well as their gene constructs against M. tuberculosis might be assessed in murine experimental models.

Namely, Balb/c mice could be immunised with the vaccine preparations by using suitable immunisation schedules. Groups of mice should receive control material (irrelevant protein or plasmid not containing the relevant insert). Antibody levels might be assayed by ELISA and Western blots while CMI elicitation could be assessed by in vitro antigen-stimulated mouse splenocyte proliferation and cytokine production [56, 57].

For protection assessment, immunised mice could be challenged i.p. by intranasal route with M. tuberculosis H37Rv strain and followed for mycobacterial organ invasion by colony-forming units enumeration (2–4 months of follow-up) or any associated mortality. The controls in these experiments would be the animals immunised with irrelevant proteins or plasmids (negative) and animals which received BCG vaccine.

The previous data on the protective effect of mAbKT4 idiotypic vaccination suggests that the recombinant KTR or Id mAbKT4 products as well as their gene constructs could be used as vaccines to control PCP. PCP could be prevented in transplant recipients or leukaemia patients before the iatrogenic induction of immunosuppressed state. HIV-infected patients could also be protected against PCP by vaccination before the full decay of their immunological response [58].

Among the many animal models that in principle could be used for examining the protection conferred by KTR and Id mAbKT4 based vaccines, the untreated rabbits at weaning and the corticosteroid-treated rats or mice should be favoured.

Spontaneous PCP develops in rabbit by days 26–34 after birth. Therefore, an active immunisation should be performed between the birth and the age of 30 days.

A potential immunisation protocol to weaning rabbits could consist in a series of subcutaneous injections of the candidate vaccine after birth. The kinetics of the induced antibody response could be established as well as their anti-Pneumocystis activity, by using in vitro tests as previously described [26, 27]. Rabbits should be sacrificed 30–40 days after birth in order to quantitate P. carinii organisms in their lungs.

Only recently, Ceré et al. (personal communication) have obtained P. carinii-free rabbit colonies as confirmed by a diagnostic PCR. These authors have succeeded in infecting P. carinii-free rabbits by intratracheal route. This model could be adopted and P. carinii-free rabbits might be immunised with KTR or Id mAbKT4 based vaccines before or after intratracheal inoculation with rabbit-derived viable, infecting parasites. Among others, this model has the main advantage that inocula can be quantitated and Pneumocystis strains may be genetically characterised.

Experimental PCP with high parasite yields can be obtained in rodents by corticosteroid administration for 9–12 weeks. Oral prednisolone or dexamethasone in drinking water are used. The protocol to evaluate the protective effect of a KT-like induced antibody response would consist in the immunisation of rodents before corticosteroid administration. Control animals should be immunised with irrelevant molecules. When the specific antibody response would be significantly high, all animals could be submitted to corticosteroid treatment for 10 weeks in order to induce PCP. Parasite counts and/or histopathological evaluation of the PCP burden should be comparatively evaluated in the two animal groups.

12 Transdisease control strategy

12.1 Production of human secretory microbicidal antibodies in transgenic plants (microbicidal antibodies)

The production of human secretory antibodies with microbicidal activity could be obtained by using transgenic plants which represent today an exclusive system to produce human secretory recombinant antibodies with predetermined specificity and in the largest quantity [59].

In particular, the genes encoding for the variable regions (VH and VL) of microbicidal murine anti-idiotypic antibodies might be amplified by PCR and inserted under the control of a promoter into vectors containing the sequences encoding the human constant regions IgA2m and kappa, respectively. The resulting constructs, together with a vector for the J-chain and the secretory component could be used for the transformation of the plant Medicago sativa by bombardment of the leaves with DNA-coated microprojectiles. Regeneration and selection of the transgenic plants could allow the evaluation of the microbicidal activity of the antibodies produced by secretion as well as the production in large quantities by squeezing and filtration of the plants.

12.2 Molecular modeling of microbicidal antibody idiotypes

It would be possible to identify the active site of KT, of the paratope of the microbicidal antibodies, and of the immunogenic idiotope of mAb KT4 in P. anomala KT and KT-mAb KT4 as well as mAb KT4-mAb K10 immunocomplexes, through X-ray crystallisation [60]. Studies of molecular modeling could allow the synthesis of the smallest molecules mimicking either the biologically active epitope of KT, and the paratope of anti-idiotypic antibodies representing its internal image or the neutralizing idiotope of mAb KT4 to be evaluated as new anti-microbial agents and new transphyletic vaccine, respectively. Analogously, the cloning of the genes coding for the variable regions of microbicidal antibodies and their sequencing should allow the identification of the regions of the paratope which could be recognised as low molecular weight molecules still displaying microbicidal activity.

12.3 Production of human recombinant microbicidal antibodies

On the basis of the occurrence of human natural microbicidal antibodies ascertained in patients infected with the sensitive microorganisms such as C. albicans, P. carinii and M. tuberculosis, the selection and characterisation of human recombinant microbicidal antibodies in the format of FAb could be obtained by human libraries representing the entire antibody repertoire [61]. The mRNA purified from the lymphocytes of the bone marrow of selected donors could be transcribed to cDNA and the genes coding for VH and VL regions amplified by PCR and cloned into the phagemid vector pComb3. The screening of the libraries could be carried out by using mAb KT4 for the selection of the positive clones which, after further amplification in a strain of E. coli, XL1 Blu, should allow the extraction and enzymatic digestion of the phagemidic DNA for the production of soluble FAb. Such FAb, after purification by affinity chromatography, could be evaluated for microbicidal properties against different sensitive pathogenic microorganisms using standardised biological methods. The cloning of the genes encoding human microbicidal FAb should allow to identify the sequences responsible for the biological activity for comparison with homologous murine microbicidal antibodies in order to produce recombinant molecules of human origin characterised by microbicidal activity.

13 Conclusions

The transphyletic anti-infectious control strategy based on the yeast killer phenomenon outlined in this paper is of potential social and economical impact being concerned with people's health. The strategy is based on conceptually new approaches for the production of antibody-derived antibiotics and transphyletic vaccines based on a natural immune response to microbial infections as well as mechanisms of biological competition evolved by microorganisms in natural habitats. Modern biotechnologies could allow a remarkable progress in the field of therapeutics and prophylaxis against microbial infections as well as the bioavailability of efficient products characterised by economicity, a prejudicial condition to face industrial needs. Last, but not the least, the proposed molecular models might represent, at the moment, the unique system for prevention and control of infectious diseases characterised by high morbidity and mortality.


The transdisease anti-infectious prevention and control strategies against diverse pathogenic microorganisms envisaged in this paper are part of a new Biotechnology research project presented to the European Community which is the direct prosecution of the collaborative work carried out within the European Concerted Action on Pneumocystis and Pneumocystosis. We would like to thank the former and latter participants for the results we have obtained and the ones that we will hopefully obtain in the future: Dr. Eduardo Dei-Cas, Dr. Jean-Charles Cailliez, Dr. Nathalie Séguy (Institut Pasteur de Lille, France), Pr. Antonio Cassone, Dr. Flavia De Bernardis (Istituto Superiore di Sanità, Rome, Italy), Dr. Ann E. Wakefield (University of Oxford, UK), Dr. Andrea Crisanti (Imperial College, London, UK), and Dr. Annick Mercenier (Institut Pasteur de Lille, France).


  1. [1].
  2. [2].
  3. [3].
  4. [4].
  5. [5].
  6. [6].
  7. [7].
  8. [8].
  9. [9].
  10. [10].
  11. [11].
  12. [12].
  13. [13].
  14. [14].
  15. [15].
  16. [16].
  17. [17].
  18. [18].
  19. [19].
  20. [20].
  21. [21].
  22. [22].
  23. [23].
  24. [24].
  25. [25].
  26. [26].
  27. [27].
  28. [28].
  29. [29].
  30. [30].
  31. [31].
  32. [32].
  33. [33].
  34. [34].
  35. [35].
  36. [36].
  37. [37].
  38. [38].
  39. [39].
  40. [40].
  41. [41].
  42. [42].
  43. [43].
  44. [44].
  45. [45].
  46. [46].
  47. [47].
  48. [48].
  49. [49].
  50. [50].
  51. [51].
  52. [52].
  53. [53].
  54. [54].
  55. [55].
  56. [56].
  57. [57].
  58. [58].
  59. [59].
  60. [60].
  61. [61].
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