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Towards third-generation whooping cough vaccines Rino Rappuoli, Mariagrazia Pizza, Audino Podda, Maria Teresa De Magistris and Luciano Nencioni To date, the most significant use of recombinant-DNA technologies has been to hyperproduce natural molecules that are difficult to obtain in large quantities by conventional methods. However, genetic manipulation can also be an efficient way to modify the properties of natural molecules in order to make them more suitable for human use. In the development of third-generation whooping cough vaccines, recombinant-DNA methods were used to remove the enzymatic activity of pertussis toxin in order to obtain a new molecule which is devoid of toxicity, and can be used for safer vaccination against this disease.

Whooping cough is a disease caused by Bordetella a Gram-negative bacterium which colonizes the upper respiratory tract of humans !. The infection starts with the adhesion of the bacteria to the cilia of the tracheal epithelial cells and is followed by a local multiplication. The many toxic substances produced cause local and systemic damage that are responsible for the clinical disease. Locally, the infection damages the respiratory epithelium and kills the ciliated cells. Systemically, one observes an increase in blood lymphocyte number, hyperinsulinemia and slight fever. The clinical disease is characterized by paroxysmal coughing, apnoea, seizures and encephalopathy. Mortality is quite common in developing countries where it can be as high as one in 100, and rare (from one in 10000 to one in 100000), but still present in Western countries. Each year there are over 60 million cases and over half a million deaths due to pertussis 2. Although pertussis can occur at any age, most of the cases occur in children, and mortality is prevalent in infants below one year of age. Antibiotic treatment is very effective in clearing the bacteria, but does not affect the course of the disease, mostly because the disease is usually diagnosed when the bacteria have already damaged the respiratory tract and released the toxins that are responsible for the severe consequences. In the absence of an effective treatment, prevention through vaccination is of extreme importance.

pertussis,

R. Rappuoli, M. Pizza, A. Podda, M. T. De Magistris and L. Nencioni are at the Sclavo Research Center, Via Fiorentina 1, 53100 Siena, Italy. TIBTECHJULY1991 (VOL9)

The first vaccine An effective vaccine against whooping cough was developed in the 1940s. This cellular vaccine is composed of whole B. pertussis cells killed by formalin and treated at 56"C for 30 min. This vaccine has virtually eliminated whooping cough and its associated mortality and morbidity in those countries where it has been used. However, the use of the cellular vaccine is very controversial because of its side effects. These can be redness, pain, induration and fever which occur in most of the vaccinees; prolonged crying, convulsions and collapse which occur with a frequency of 0.1-1%. In addition to these mild reactions, severe side effects such as brain damage and death have in the past been associated with pertussis vaccination3. Although these reactions do not seem to be due to the vaccination4, the concern about them has decreased the acceptance of the vaccine in many Western countries. For example, in the early 1970s, a few cases of neurological damage associated with pertussis vaccination were recorded in the UK. These were widely publicized by the media and vaccine acceptance decreased from 90% to 30%. As a consequence, the incidence of pertussis cases increased greatly, reaching rates of over 100000 reported cases per year and several dozen deaths due to disease were recorded s. The conclusion was that the risks associated with vaccination are minimal compared to those associated with the disease and the World Health Organization (WHO) recommended continuing vaccination with the cellular vaccine, and focusing the research on the development of new vaccines devoid of side effects. Since then, uptake of vaccination has increased up to 70% in the UK, while it is still very low or absent ~) 1991, ElsevierSciencePublishersLtd (UK) 0167- 9430/91/$2.00

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reviews LPS Proteins responsible for the side effects caused by the cellular vaccine9. These two findings raised the possibility that pertussis toxin could be the antigen required for protection and also the toxin responsible for the i severe side effects of the cellular ~ili Second-generation vaccines vaccine. Subsequently, most of the -~!~ When modem technologies are used to analyse antigens listed jn Table 1 were ,~.~ the composition of the cellular vaccine developed in purified, chemically detoxified and the 1940s, it is found to be composed of many tested as vaccines in the mouse intradifferent molecules including proteins, polysac- perebral challenge assay, a test .... charides, lipids and nucleic adds. The vaccine is which correlates with the protective also overloaded with lipopolysaccharide, a com- efficacy of the cellular vaccine in ponent known to be toxic, and that by itself can children. In this assay, pertussis account for many of the side effects (Fig. 1). Some toxin was the only antigen able to of the components of the cellular vaccine are induce protection, while other mob known to be important for the virulence of B. ecules such as FHA and the 69 kDa pertussis (Table 1) and, since they are antigenic, are outer-membrane protein, 69K (also considered potentially useful for vaccine develop- named pertactin), although not proment; most of the other components of the cellular tective on their own, were able to Figure 1 vaccine are useless in this respect and many are increase the potency of the vaccines toxic. In order to develop a new vaccine, the containing chemically detoxified SDS-polyac~amide gels of a antigens that confer protection need to be identified pertussis toxin. On the basis of these sample of cellular vaccine stained and separated from the others. A first step towards observations, a number of acellular with silver stain to detect proteins this goal was achieved by Y. Sato who purified two vaccines were developed and tested or lipopolysaccharides (LPS), proteins (pertussis toxin [PT] and filamentous for safety and efficacy in animal hemagghtinin [FHA]), from the culture super- models (Table 2). As shown in Table 2, all the natant of B. pertussis, inactivated them with for- vaccines proposed contain pertussis toxin detoximaldehyde and developed the first acellular vaccine fled by different chemical methods, either alone or which has been used in Japan since 19816. A more associated with one or more of the other antigens. detailed analysis, to identify the antigens respon- Two of the vaccines shown in Table 2 (PT and PT+ sible for protection and side effects, required the development of the genetics of B. pertussis and of new animal models. In the laboratory ofS. Falkow, Table 1. Virulence factors of B. pertussis A Weiss constructed a number of mutants of Virulence factor Role in pathogenesis B. pertussis defective of individual virulence factors Pill and showed that only pertussis toxin and adenylate Adhesion Filamentous hemagglutinin (FHA) cydase are strictly required for the virulence of 69 kDa outer-membrane protein (69K) B. pertussis 7"s. This finding implied that immunity against one of these antigens may be enough for Avoiding host defences Pertussistoxin (PT) protection. Adenylate cyclase (Adc) Invasion Dermonecytotic toxin (Dnt) L. Steinman developed an assay in mice showing Hemolysin (Hly) that minute traces of active pertussis toxin were Tracheal cytotoxin (Tc) able to induce an anaphylactic syndrome and this Toxicity Lipopolysaccharide (LPS) suggested that a similar mechanism could be

in other countries such as Italy, Germany and Sweden. In the USA, where vaccination has been widely accepted and never discontinued, the morbidity and mortality due to whooping cough is absent. However, even in this country a safer vaccine is eagerly anticipated.

Table 2. Proposed acellular vaccines against pertussis Vaccine composition

Method of PT detoxiflcation

Laboratory studies or clinical trials

PT PT PT PT + PT + PT + PT + PT PT +

Formaldehyde Hydrogen peroxide Tetranitromethane Formaldehyde or glutaraldehyde Formaldehyde Formaldehyde Formaldehyde Genetic manipulation of PT gene Genetic manipulation of PT gene

Phase III clinical trials Phase II clinical trials Phase II clinical trials Phase Ill clinical trials Phase II clinical trials Laboratory studies Laboratory studies Phase II clinical trials Phase II clinical trials

FHA FHA + P i l i 69K FHA + 69K FHA + 69K

Refs 6,10

27 28 6, 10, 29 3O

a

a

23, 25 23, 25 and a

a Unpublished data

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FHA) have been tested in large-scale clinical trials carried out in Sweden !°. Both vaccines offered protection from severe disease with an efficacy similar to that of the cellular vaccine. However, during the cSnical trials the pertussis toxin showed some reversion to toxicitylk The conclusion was that pertussis toxin, either alone or combined with other antigens, can be an effective vaccine against whooping cough. Nevertheless, in order to avoid the risks of side effects, better methods of detoxification were required. To achieve this, many laboratories investigated chemical methods as alternatives to the formalin treatment (Table 2). In our laboratory we decided to approach the same problem using recombinant-DNA technology.

/.

Failure o f d o n e d PT genes to yield efficacious vaccine Pertussis toxin (PT) is a complex bacterial protein toxin composed of five non-covalently-linked subunits named $1-$5 (Fig. 2). $1 is an enzyme which intoxicates eukaryotic cells by ADP-ribosylating their GTP-binding proteins. Subunits $2, $3, $4 and $5, present in a 1:1:2:1 ratio, bind the receptors on the surface of eukaryotic cells and facilitate the translocation of the $1 subunit across the cellular membrane so that it can reach its target proteins 12. In order to clone the genes coding for PT, we purified the five individual subunits, determined their amino-terminal amino acid sequence and used this to design oSgonucleotides which were then used to identify the PT clones in a

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Figure 4 Recombinant fragments of the S| subunit containing amino- and carboxy-terminal deletions which were used to map the protective epitope recognized by the monoclonal antibodies,the T-cell epitopes recognized by the human T-cell clones, and the minimal region required for enzymatic activity. Below the map of the fragments we report a scheme of the three regions (a, b and c) required to form the conformational protective epitope (B-cell epitope) and the regions where we mapped the T-cell epitopes. TIBTECHJULY1991(VOL9)

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b

Rgure 5 Schematic representation of the protective epitope of the SI subunit which is present only in (a) the nativesubunitin the presenceof subunits $2-$5, but is not presentin (b) the recombinant,enzymaticallyactive S1 subunit.

library of B. pertussis DNA. The nudeotide sequence revealed that the PT genes are organized as an operon as shown in Fig. 313. Similar work was also carded out at the Rocky Mountains laboratory by Jerry Keith and Camille Locht 14. Initial attempts to obtain a recombinant vaccine by expressing the PT operon failed because Escherichia coil was unable to express the cloned genes. As an alternative approach, the five subunits were expressed individually, either as natural or fusion proteins, and good yields of the recombinant proteins were obtained. The recombinant $1 subunit retained full enzymatic activity. Although these recombinant subunits were unable to assemble into the holotoxin, they were expected to be efficacious vaccines because, at least in the case of the $1 subunit, it had been shown that monoclonal antibodies (mAbs) against the native protein were able to protect mice from the infection with virulent B. pertussis. However, it was found that the recombinant subunits were very immunogenic but unable to induce protective immunity; in other words, they induced a lot of antibodies which recognized the PT subunits in a Western blot but did not neutralize the toxin in vitro nor protect mice from infection with virulent B. penussis is. This finding implied that immunization with the native, detoxified PT or with the recombinant subunits induced antibodies with similar specificities and titres (as measured in a Western blot), but which were functionally very different. In order to understand this discrepancy, we decided to map the protective epitopes recognized by the anti-S1 mAbs. To do so, we collected seven different mAbs, and tested them for their reactivity with a series of recombinant $1 subunits bearing carboxy- and aminoterminal deletions (Fig. 4). All seven protective mAbs were found to recognize a single epitope composed of three regions which are noncontiguous in the primary structure of the $1 subunit, but are close together in the threedimensional structure of the assembled toxin 16. By radioimmunoassay, it was found that this epitope is present in the native PT but absent in the recombinant subunit. This fmding (shown schematically in Fig. 5) provided a reasonable explanation for the

Rgure 6 Gene replacement in Bordetella pertussis. A schematic representation of the method used to replacethe chromosomalgene codingfor pertussistoxin (TOX),The DNA regions which flank the pertussis toxin operon were clonedat eachside of the gene codingfor the kanamycin resistance (KANr) in the plasmid PRTPZa2 which is not able to replicate in Bordetella. The plasmid was then conjugated in Bordetella and upon selection with kanamycin only those bacteria which had integratedthe plasmid into the chromosome were selected. A new selection was then applied to favour the second homologous recombination which causes the loss of the PToperonend replacesit with the kanamycin gene. Then, the kanamycin gene was replaced with a DNA fragment coding for the mutated PT genes repeatingthe procedure described above.

failure to obtain protective immunity using a recombinant $1 subunit and suggested that alternative strategies were necessary to develop an efficacious vaccine.

Construction oggenedcally detoxilied pertmsis toxin molecules In theory, the best vaccine against whooping cough should contain a PT that is devoid of TIBTECHJULY1991(VOL9)

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reviews Table 3. In vivo and in vitro properties of purified native PT and genetically detoxified PT for comparison Native PT

Mutant PT-gK/129G

(ng m1-1) (lag) (lag mouse-1) (tag mouse-]) (lag mouse-l) (lag mouse-]) (tag I',g-~)

0.005 0.001 0.1-0.5 0.02 0.04 5000 >20 >50 >50 >7.5 >25 >1500

T-cell mitogenicity

(Lagm1-1)

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(lag well-1)

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[Ka(L tool-l)] a 2.4 x 10s [Ka(L tool-l)] a 2.0 x 10 lo

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enzymatic activity but maintains the appropriate Band T-cell epitopes. Since we had already mapped the protective B-cell epitope, we focused our research on mapping the T-cell epitopes and identifying amino acids that are important for the enzymatic activity. To map the T-cell epitopes, we obtained more than 20 human T-cell clones specific for PT. Eighty-five percent of the clones were found to be specific for the $1 subunit, indicating that this subunit contains the immunodominant epitopes both for B- and T-lymphocytes. The epitopes recognized by the T-cell clones were then located using the recombinant $1 molecules with deletions at the amino- and carboxy-termini (shown in Fig. 4) and finally mapped at the amino-acid level using synthetic peptides 17. The $1 subunit was shown to contain three major T-cell epitopes (see Fig. 4). Among these, the one mapping in the region 30-41 was immunodominant. To map the amino acids important for enzymatic activity, we first tested the recombinant fragments containing amino- and carboxy-terminal deletions (shown in Fig. 4). We found that the fragment containing amino acids 2-180 was the minimal polypeptide capable of fuU enzymatic activity. The region 2-180 was mutagenized in vitro, using sequence homologies with other bacterial toxins, computer-modelling predictions and guesses in order to identify the essential amino acids. We and other laboratories identified several amino acids whose substitution abolished the enzymatic activity of the $1 subunit is. These included Arg9, Arg13, Trp26, His35, Arg58 and Glu129 (Refs 1%21). The genes containing mutations for these amino acids were then introduced into the chromosome of B. pertussis using homologous recombination between the chromosomal gene and sequences introduced on a plasmid unable to replicate in B. pertussis22 (see Fig. 6). The B. pertussis strains TIBTECHJULY1991(VOL9)

containing one of the above amino acid substitutions in the $1 subunit produced PT molecules which had a toxicity 10-1000 times lower than the wild-type pertussis toxin. However, this toxicity was still too high for use in a vaccine. In order to reduce the toxicity further, two of the above amino acids were substituted in a single $1 subunit, thus obtaining several PT double-mutant molecules which, in the chinese hamster ovary (CHO) cell clustering assay, had a toxicity lower than 0.0001% compared with the wild-type toxin 23. These included mutations in positions 9/129, 13/129 and 26/129. In particular, Glu129 was replaced by Gly, Arg9 with Lys, Arg13 with Leu and Trp26 with Leu. These mutants did not show any of the toxic properties typical of PT such as lymphocytosis, histamine sensitivity, potentiation of anaphylaxis (Table 3), but maintained intact the B- and T-cell epitopes and the protective activity of the wild-type toxin. In fact, these mutants were able to compete with 12SI-labded PT as efficiently as wild-type PT for binding to a protective mAb against the $1 subunit and for binding to a polyclonal antibody against the holotoxin. Similarly, they were recognized by the human T-cell clones specific for the $1 subunit, confirming that the mutations introduced had not altered the immunological properties of the new molecules. Furthermore, they maintained the natural non-toxic properties of PT such as the ability to agglutinate red blood cells, and the mitogenicity for T cells (Table 3). When tested for the ability to induce toxin neutralizing antibodies, two doses of 3 ~g of these mutants were found to induce toxin neutralizing titer of >1/1280, a value higher than that generally obtained with chemically detoxified PT. Finally, the same mutants were able to protect mice from the intracerebral challenge with virulent B. pertussis in a dose-dependent fashion24. Since this last test correlated with the efficacy in humans of the cellular vaccine, we concluded that the genetically detoxified pertussis toxin molecules were ideal candidates for the development of acellular pertussis vaccines. Clinical tests with the third-generation vaccines After extensive studies in vitro and in animal models to demonstrate the safety and the immunogenicity of the genetically detoxified pertussis toxin molecules, we tested the new molecules as vaccines in adult volunteers. We used two vaccines; one containing only 15 gg of the mutant PT-9K/129G, the other containing 7.5 ~g of the mutant PT-9K/129G, 10 l~g of FHA and 10lag of the 69K protein (Fig. 7). Both vaccines were safe and more immunogenic than the chemically detoxified PT molecules 25. These results encouraged us to proceed and test the vaccines in infants (i.e. the target population for vaccination against pertussis). A phase II clinical

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reviews PT-9K/129G 69K

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intact all the epitopes which are normally modified by chemical treatment. The results obtained so far show that the third-generation vaccines against whooping cough have solved these problems that are associated with the cellular and secondgeneration vaccines. They are likely, therefore, to become of general use in the near future.

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Figure 7 SDS-polyacrylamide gel showing the composition of the thirdgeneration pertussis vaccines. These contain highly purified FHA and 69K and a highly purified PT analogue that has been detoxified by genetic manipulation. A comparison to Fig. 1 gives an idea of the progress made from first to third-generation vaccines.

trial involving more than 250 infants was designed so that each infant would receive three doses of the vaccine. Preliminary results of this clinical trial confirm the safety of the new vaccine and its high immunogenicity. Conclusions From the very beginning of the history of vaccination, chemical detoxification has been considered the best way to obtain safe and efficacious vaccines. However, chemical detoxification has its disadvantages since it requires the handling of very dangerous material. Moreover, each lot needs to be tested carefully for the detoxification, and there is a risk of reversion to toxicity. Furthermore, the antigens obtained are of low purity and their antigenic properties have been heavily modified by the chemical treatment 26. With the advent of biotechnology, molecular genetics has provided new tools to inactivate toxins for vaccine use. The story reported in this review shows that it is possible to modify one or more codons in their genes and obtain nontoxic molecules which do not need chemical treatment, can be purified to homogeneity and for which the risk of reversion to toxicity is absent. These molecules are not only safer but also superior immunogens since they maintain

1 Moxon, E. R. and Rappuoli, R. (1990) Lancet i. 1324--1329 2 Muller, A. S., Leeuwenburg, J. and Pratt, D. S. (1986) Bull. WHO 64, 321-331 3 Miller, D. L., Ross, E. M., Aldersladc, R., Bellman, M. H. and Rawson, N. S. B. (1981) Br. Med.J. 282, 1595-1599 4 Cherry, J. D. (1990)3. Am. Med. Assoc. 263, 1679-1680 5 Cherry, J. D., Brandl, P. A., Golden, G. S. and Karzon, D. T. (1988) Pediatrics81,933-934 6 Sato, Y., Kimura, M. and Fukumi, H. (1984) Lancet i, 122-126 7 Weiss, A. A., Hewlett, E. L., Myers, G. A. and Falkow, S. (1983) h!fect, hnmun. 42, 33-41 8 Weiss, A. A., Hewlett, E. U, Myers, G. A. and Falkow, S. (1984)J. h!fect. Dis 150, 219--222 9 Steinman, L., Weiss, A., Addman, N., Li~Ji, M., Zuniga, R., Ochlert, J., Hewlett, E. and Falkow, S. (1985) Proc. Natl Acad. Sci. USA 82, 8733-8736 10 Ad hoc group for the study of permssis vacdne (1988) Lancet i, 955-960 11 Storsaeter, J., Hallander, H., Farrington, C. P., Olin, P., Mollby, R. and Miller, E. (1990) Vacchze8, 457-461 12 Tamura, M., Nogimori, K., Murai, S.. Yajima, M., lto, K., Katada, T., Ui, M. and lshii, S. (1982) Biochemistry 21, 5516-5522 13 Nicosia, A., Perugini, M., Franzini, C., Casagli, M. C., Borri, M. G., Antoni, G., Almoni, M., Ncri, P., Ratti, G. and Rappuoli, R. (1986) Proc. Nail Acad. Sci. USA 83, 4631-4635 14 Locht, C. and Keith, J. M. (1986) Science 232, 1258--1264 15 Nicosia, A., Bartolini, A., Perugini, M. and Rappuoli, R. (1987) b#'ea, lmmun. 55, 963-967 16 Bartoloni, A., Pizza, M., Bigio, M., Nucci, D., Ashworth, L. A., Irons, L. I., Robinson, A., Burns, D., Manclark, C., Sato, H. and Rappuoli, R. (1988) Bio/Teclmology 6, 709-712 17 De Magistris, M, T., Romano, M., Bartoloni, A., Rappuoli, R. and Tagliabue, A. (1989)J. Exp. Med. 169, 1519-1532 18 Pizza, M., Bartoloni, A., Prugnola, A., Silvestri, S. and Rappuoli, R. (1988) Proc. Nail Acad. Sci. USA 85, 7521-7525 19 Barbieri, J. T. and Cortina, G. (1988) b~fect, lmmun. 56, 1934-1941 20 Burnette, W. N., Cieplak, W., Mar, V. L., Kaljot, K. T., Sato, H. and Keith, J. M. (1988) Science 242, 72-74 21 Loosmore, S. M., Zealey, G. R., Boux, H. A., Cockle, S. A., Radika, K., Fahim, R. E. F., Zobrist, G. J., Yacoob, R. K., Chong, P. C. S., Yao, F. L. and Klein, M. H. (1990). b!fect. hnmun. 58, 3653-3662 22 Stibitz, S., Black, W. and Falkow, S. (1986) Gene 50, 133-140 23 Pizza, M G., Covacci, A., Bartoloni. A., Perugini, M., Nencioni, L., De Magistris, M. T., Villa, L., Nucci, D., Manetti, R., Bugnoli, M., Giovannoni, F., Oliveri, R., Barbieri, J., Sato, H. and Rappuoli, R. (1989) Science 246, 497-500 Nencioni, L., Pizza, M., Bugnoli, M., De Magistris, T., Di Tommaso, A., Giovannoni, F., Manetti, R., Marsili, I., Matteucci, G., Nucci, D., Olivieri, R., Pileri, P., Presentini, R., Villa, L., Kreefienberg, J, G., Silvestri, S., Tagliabue, A. and Rappuoli, R. (1990) b!fea, hnm,n. 58, 1308-1315 25 Podda, A.. Nencioni, L., De Magistris, M. T., Di Tommaso, A., Boss, P., Nuti, S., Pileri, P., Peppoloni, S., Bugnoli, M., Ruggiero, P., Marsili, !., D'Errico, A., Tagliabue, A. and Rappuoli, R. (1990)J. Exp. Med. 172, 861-868 TIBTECHJULY1991 (VOL9)

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reviews 26 Nencioni, L., Vo~Fiai, G., Peppoloni, S., De Magistris, M. T., Marsili, i. and Rappuoli, R. (1991) hzfect, hnmun. 59, 625-630 27 Sekura, R. R. D., Zhang, Y., Roberson, R., Acton, D., Trollfors, B., Tolson, N., Shiloach, J., Bryla, D., Muir-Nash, J., Koeller, D., Scheerson, R. and Robbins, J. B. (1988) J. Pediatr. 113, 806-813 28 Winberry, L., Walker, R., Cohen, N., Todd, C., Sentissi, A., Siber, G. (1988)Abstracts ofhzternational Workshopof B. pertussis (Keith, J., ed.), Hamilton Rocky Mountains Laboratory 29 Edwards, K. M., Bradley, R. B., Decker, M. D., Palmer, S. P., Van Saraga, T., Taylor, J. C., Dupont, W. D., Hager, C. C. and Wright, P. F. (1989).]. hzfect. Dis. 160, 832-837 30 Rutter, D. A., Ashworth, L. A. E., Day, A., Funnel, S., Lovell, F. and Robinson, A. (1987) Vaccine6, 29-32

Common principles in protein

folding and antigen presentation Shan Lu, Victor E. Reyes, Rochelle R. Torgerson, Robert A. Lew and Robert E. Humphreys The regular recurrence of hydrophobic amino acid residues along a peptide sequence determines the formation of a longitudinal hydrophobic strip when the peptide forms an o~-helix.An understanding of the ways this may affect both folding of nascent proteins and antigen presentation should facilitate vaccine and therapeutics design. Formation of an 0c-helix in a protein situated against a hydrophobic surface is determined by the existence of hydrophobic amino acids recurring regularly at specific positions along the peptide chain. These residues form a longitudinal hydrophobic strip when the sequence is coiled as an 0~-helix. This principle appears to govern both formation of helices in folding intenaediates of nascent proteins and scavenging of T-cell-presented epitopes in proteolytic fiagments of antigenic protein. The mechanisms which regulate digestion of antigenic proteins into specific fragments, and selection of only some fragments for transfer to major histocompatibility complex (MHC) molecules are unclear. We hypothesize that consensus sequences (in or around the sequence which actually fits into the antigenbinding site of MHC molecules) are involved. If such consensus sequences are found, an understanding of the biophysical or chemical mechanisms by which they act will permit rational

engineering of such sequences. One might exploit this idea to engineer potency and range of MHC restriction of vaccines, or to alter immunogenicity of therapeutic proteins.

Helical folding of antigenic peptides

DeLisi and Berzofsky noted that many T-cellpresented peptides are amphipathic helices 1. Kaiser and K~zdy had observed that amphiphilic peptides can coil on contact with lipid membranes 2. The progressive anchoring of hydrophobic side chains into a lipid bilayer at successive turns of a peptide coiling into a helix might co-operate with hydrogen bonding along the pepddyl backbone to stabilize the growing helix (Fig. 1). Such membrane adsorption could be a step in transferring peptides to MHC molecules3. Such recurrent hydrophobic residues would form a longitudinal hydrophobic strip along the helix4-7. During antigen processing, binding of such peptides to the wall of a vesicle could lead to selective transfer of some digested peptides to a second compartment after expulsior~ of soluble contents S. Lu, V. E. Reyes, R. R. Torgerson, R. A. Lew and R. E. to a lysosome-bound vesicles;9. Alternatively, the Humphreys are at the Department of Pharmacology, University hydrophobic strip of such an adsorbed helix could of Massachusetts Medical School, 55 Lake Avenue North, promote its binding to the hydrophobic floor of Worcester, MA 01655, USA. either a scavenging/transfer molecule or a MHC

TIBTECHJULY 1991 (VOL9)

~) 1991, ElsevierScience Publist~ersLtd (U;q 0167-9430/91/$2,00

Towards third-generation whooping cough vaccines.

To date, the most significant use of recombinant-DNA technologies has been to hyperproduce natural molecules that are difficult to obtain in large qua...
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