© 1997 Nature Publishing Group http://www.nature.com/naturemedicine COMMENTARY• Rational design of vaccines The recombinant pertussis vaccine induces early and long-lasting protection 2 A few years ago Sir Gustav Nossal re­ lular pertussis vaccines • Most of these vac­ ported in a book that during the fifties RINO RAPPUOLI cines contain a form of PT detoxified by the Nobel Prize winner Joshua Leder berg chemical treatment (that is, by glutaralde­ "frequently voiced the view that the presently available vac­ hyde, formaldehyde, hydrogen peroxide), a procedure that was cines were a scandal." He questioned "how could we inject our developed at the beginning of the century to detoxify other children with the horrible mixture of substances present in toxins such as diphtheria and tetanus'. The vaccine here de­ killed H. pertussis," and pushed for the use of modern science to scribed is unique, because it contains a mutated, nontoxic form develop "defined molecular vaccines'n. of pertussis toxin that is genetically detoxified. The mutant Some forty years later the challenge proposed by Joshua toxin is produced by an engineered strain of Bordetella pertussis Lederberg has been successfully taken up and his dream of see­ containing the pertussis toxin gene that has been modified by ing defined molecular vaccines has been fulfilled (see Table 1 site-directed mutagenesis in order to introduce two amino acid for the key steps). The substitutions (Arg9~Lys and most advanced scientific Table 1 Key steps of the development Glu129~Gly), in the active site of knowledge has been ap- of the recombinant pertussis vaccine the enzyme. These amino acid plied to design rationally changes render inactive the cat­ and to develop a geneti- 1984 The project starts alytic site and make the molecule cally engineered vaccine 1985 Cloning and sequencing of the pertussis toxin gene nontoxic' (Fig. 1). Therefore, the against pertussis that is 1987 Identification of the key amino acids to be changed by mutant molecule can be used in molecularly defined and mutagenesis to make a nontoxic derivative of pertussis toxin the vaccine without further chem­ 1988 Sequence of the gene coding for filamentous hemagglutinin that confers maximal pro- ical detoxification. This approach 1989 Construction of a Bordetello pertussis strain producing a tection from disease by nontoxic mutant of pertussis toxin offers several advantages. First, using low amounts of Phase I clinical trials the mutant PT exhibits none of antigens, thus providing a 1990 Phase II trials with acellular pertussis vaccine, alone (aP) or the toxic properties of the wild­ safe product. combined with diphtheria and tetanus (DTaP) type toxin, while retaining all This vaccine represents 1992 Beginning of the phase Ill efficacy trial with a DTaP vaccine other biological activities of the the first example of a mol- 1993 Approval and marketing of the aP vaccine in Italy molecule. Second, the genetically 19 ecule constructed by ra- 95 End of the phase Ill efficacy trial, approval and marketing of the engineered mutant PT is expected DTaP vaccine in Italy tional drug design that to retain the correct conformation 1996-97 Filing of the product license application worldwide has been proven effica­ of the wild-type toxin, and hence cious in a clinical trial, the conformational (neutralizing) showing that rational engineering may indeed be used to im­ epitopes known to exist in the five subunits of the toxin. In prove the effects and the safety of vaccines and possibly drugs. fact, a recent study using a panel of monoclonal antibodies (see Table 2) has shown that toxin-neutralizing epitopes were well The genetically engineered pertussis toxin: conserved in the genetically detoxified PT; however, these epi­ from the laboratory to a vaccine ... topes were destroyed by the chemical detoxification of PT, Pertussis toxin (PT) represents the key antigen in modern acel- which is usually used for vaccine production. Fig. 1 Schematic representation of the cavity containing the active site of pertussis toxin and rationale for genetic detoxification. The toxic activity of pertussis toxin is mediated by the ADP-ribosyl­ transferase ezymatic activity. During this reaction, the ADP-ribose group of NAD' is transferred to target proteins of eukaryotic cells. The active site of the enzyme is formed by a ~ strand and an a helix that make the floor and the ceiling of the cavity, and it contains two amino acids (Arg9 and Glul29) that are essential for catalysis. Following mutagenesis of the PT gene, to replace Arg9 with Lys and Glul29 with Gly, the mutant B. pertussis strain synthesizes a PT molecule that is enzymatically inactive and therefore nontoxic. The mutant PT molecule is the basis of the recombinant pertussis vaccine. 374 NATURE MEDICINE • VOLUME 3 • NUMBER 4 • APRIL 1997 © 1997 Nature Publishing Group http://www.nature.com/naturemedicine • COMMENTARY Table 2 Recognition of detoxified pertussis toxin fewer episodes of pertussis than did the recipients of by toxin-neutralizing monoclonal antibodies the vaccine containing chemically detoxified PT, in __________::.__ _____ -=.______________ -l the period from the first immunization to 1 month mAb PT specificity Wild-type PT* Genetically Chemically detoxified PT after the third immunization (Incomplete immu­ detoxified PT (formaldehyde) nization in Fig. 3), and in the period from 17 to 26 63.1 G9 51 1.00 0.86 <0.03 months after the third immunization (stage II in Fig. 63 .2 E3 51 1.00 1.07 <0.03 3). The protection induced by the two vaccines was 63.2 G12 51 1.00 0.78 <0.02 21.3 D12 B oligomer 1.00 1.10 <0.09 similar during the period from 1 to 17 months after 21.3 D9 52 + 53 1.00 1.36 <0.23 the third immunization (stage I in Fig. 3). 21 .3 Dll 52+ 53 1.00 1.51 <0.32 Immunogenicity studies showed that in spite of 47.4 53 + 54 1.00 3.84 <0.20 the lower antigen content, subjects who received ...:...;__:_20.6 _______53+ 54 _ ______________1.00 1.17 0_ ._18____ the vaccine containing the recombinant pertussis *Ra tio of soluble toxin to toxoid required for inhibition of binding of monoclonal antibodies toxin (composed of 5, 2.5 and 2.5 Jlg of PT, FHA and to solid-phase PT (ref. 10). PRN, respectively), had a higher antibody response ---'---------------=-=----::::::=======- against the PT component than those who received the vaccine containing chemically detoxified PT ... with improved immunogenicity ... (composed of 25, 25 and 8 Jlg of PT, FHA and PRN, respec­ In the final formulation, the vaccine contains the mutant PT tively), while, as expected from the higher antigen content, the (5 Jlg/dose), filamentous hemagglutinin (FHA; 2.5 Jlg/dose) and antibody response to FHA and PRN was higher for the latter Pertactin (PRN; 2.5 Jlg/dose). This vaccine has now been tested vaccine. in several phase II clinical trials. Valuable information was ob­ The vaccine containing the genetically detoxified PT, in spite tained from a trial performed by the National Institute of of having one-sixth as much protein, induced an earlier and Allergy and Infectious Diseases (NIAID) in the USA, where the longer lasting protection from disease. As the only difference safety and immunogenicity of this vaccine were compared with between the two vaccines resides in the antigenic quality of the those of eleven vaccines containing chemically detoxified PT. PT antigen, it is very likely that the observed superior protec­ Vaccines containing the recombinant PT induced an anti-PT tive efficacy of the vaccine containing the genetically detoxi­ response 10--20 times as high (on the basis of weight) as that fied PT is due to the PT antigen, which, in spite of being present from vaccines containing chemically inactivated PTs and had a in lower quantity, induces a better immune response. steeper dose-response curve (Fig. 2)"'. A qualitatively different immune response might explain why only during stage I, in coincidence with the very short ... and efficacy peak of the maximal antibody response (shown as a green The vaccine was tested in a double-blind, placebo-controlled curve in Fig. 3), the higher quantity of the chemically inacti­ efficacy trial performed in Italy, involving approximately vated PT (25 Jlg/dose) was able to induce a level of immunity 16,000 infants. The trial was sponsored by NIAID and per­ that could compensate for the antigenic quality of the geneti­ formed by the Istituto Superiore di Sanita in Rome. The results cally inactivated PT (5 Jlg/dose). In marked contrast, the low­ showed that the vaccine containing the genetically detoxified dose recombinant PT was able to protect better from disease PT was very efficacious (84o/o) in protecting from disease, and during the period of incomplete immunization and for an ex­ during the first 30 months of observation, the subjects had tended time after the third immunization (stage II), when the fewer pertussis cases (55 cases) than those administered an effi­ antibody titers are low and the quality of the memory response cacious acellular pertussis vaccine containing the same pertus­ is essential for protection. sis antigens, but prepared by conventional chemical The success of vaccination using recombinant PT is an example inactivation (81 cases) [ref. 6, 7 and Greco, D. et al., Int. of the importance of the quality of the immune response. Congress for Infectious Diseases, Abstr. (1996)]. Hence immunization may be improved by using high-quality A close look at the results of the trial revealed that recipients antigens that can be delivered in low doses and with fewer im­ of the vaccine containing genetically detoxified PT experienced munizations, which is preferable to the high doses and multi- 200.--------------------------, Detoxified PT: I=' 150 ::E A Genetically 8 • Chemically c~ 0 ~ 100 ~ >- '0 0 Fig.