PROFILE

Profile of Rino Rappuoli ino Rappuoli grew up in the shadow of what he describes as a testament to the devastating impact of infectious disease: Rthe unfinished wall of the Cathe- dral in Siena, . When the plague hit the city in 1348, it slashed the popula- tion from 100,000 to 30,000. ‘‘It basically shut down one of the most powerful economies of the time, and that momen- tum was lost forever. I see it as an ex- ample of what could happen today with pandemic ,’’ says Rappuoli, whose current research focuses on devel- oping a for avian influenza. Rappuoli, currently the Global Head of Research for Vaccines & Diagnostics (Siena, Italy), was elected as a foreign associate of the National Academy of Sciences in 2005. He has spent his career developing vaccines for pertussis, meningitis, and Helicobacter pylori and is jointly responsible for engi- neering the carrier protein used in many conjugate vaccines. He is credited with launching the field of reverse vaccinology, the first fruits of which are revealed in his Inaugural Article in this issue of PNAS Rino Rappuoli (1), where he describes a universal vaccine for serogroup B meningococcus. fellowship at Siena’s Sclavo Research to Alwin Pappenheimer, who became a Bucolic Beginnings Center, the Italian vaccine institute that mentor to Rappuoli. ‘‘He was one of Rappuoli was born in 1952, in Radicofani, had been developing and producing vac- the fathers of and immun- Italy, a village 40 miles south of Siena. cines for almost a century. ology....Heisanother person who really When he was 11, his family moved closer shaped my career,’’ says Rappuoli. Microbiology in America to Siena, enabling Rappuoli to attend high At Harvard Medical School, Rappuoli school in the city. He spent weekends and Almost immediately after joining the joined Murphy and Pappenheimer in summers helping his father produce Chi- institute, Rappuoli left to take on a their search for a new diphtheria vac- anti, the region’s signature red wine. As project in the . He knew cine. The project stemmed from work college approached, Rappuoli was torn that to work at the cutting edge of vac- Pappenheimer had done in 1972 (2, 3). between the desires to study poetry or cine science he needed to learn new Pappenheimer had mutagenized the science. ‘‘I chose science,’’ says Rappuoli. techniques in molecular biology and ge- diphtheria toxin gene and isolated a mu- ‘‘The moon landing and the sense of im- netic engineering. In 1979, he spent four tant with a single amino acid change. pending scientific revolution probably in- pivotal months at The Rockefeller Uni- This minute tweak rendered the mole- fluenced my thinking.’’ versity (New York, NY) as a visiting cule, called CRM197, nontoxic and ideal Rappuoli pursued his undergraduate scientist in the laboratory of Emil as a diphtheria vaccine. ‘‘This was a big studies at the University of Siena but Gotschlich, who pioneered meningococ- change in thinking because instead of yearned to experience science outside of cal vaccine studies in the 1960s. ‘‘He is using chemicals to detoxify the toxin, Italy. Wanderlust led him to Washington probably one of the smartest scientists you could modify the gene, and the bac- University in St. Louis, MO, where he I’ve ever met and probably the person teria would produce a molecule you studied bacterial mutagenesis during the who had the greatest impact on me,’’ wanted already to go for you. It is per- summer vacation after his third year of says Rappuoli. ‘‘He would just tell me haps the first example of rational design college. More important than the re- about science . . . how to approach the of natural molecules to yield efficient search, however, were his observations of problem and spend a lot of time teach- therapeutics or vaccines,’’ says Rappuoli. ing me. Sometimes you meet a person the bountiful conditions American scien- Conjugate Vaccines tists enjoyed. ‘‘In Italy, the theoretical who changes the way you think.’’ training was good, but the technology gap In 1980, Rappuoli spent a year at Har- In 1981, Rappuoli returned to Sclavo Re- was huge,’’ says Rappuoli, ‘‘and molecular vard Medical School (Boston, MA) in the search Center in Italy and launched his biology was 5 or 6 years behind the laboratory of John Murphy, who worked own laboratory. He continued his collabo- United States.’’ on Corynebacterium diphtheriae, which ration with Murphy and Pappenheimer After graduation, Rappuoli remained causes diphtheria. Murphy introduced

at the University of Siena, where he Rappuoli to bacterial genetics and the This is a Profile of a recently elected member of the National earned a Ph.D. in biological sciences for world of bacterial toxins, and he helped Academy of Sciences to accompany the member’s Inaugural his NMR studies of proteins and tissue Rappuoli learn new techniques in microbi- Article on page 10834. membranes. In 1978, he was offered a ology. Murphy also introduced Rappuoli © 2006 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0604892103 PNAS ͉ July 18, 2006 ͉ vol. 103 ͉ no. 29 ͉ 10831–10833 Downloaded by guest on September 28, 2021 and was charged with mass-producing States and Europe to shift from use of the cloned the H. pylori toxin, identified the CRM197. He succeeded in 1982, using old to the new acellular mechanism of action, and made a vac- classical genetics, but CRM197 was never one. ‘‘That was a milestone in vaccination cine that went into clinical trials. But by used for a diphtheria vaccine. The current because it was a switch from traditional the end of Phase I studies, commercial diphtheria vaccine was developed in and whole-cell killed-bacterial vaccines to interest in the vaccine was low. has been in use since 1924. ‘‘That’s the more sophisticated, modern purified vac- Although the work did not yield a vaccine that we still use today,’’ says Rap- cines,’’ Rappuoli says. vaccine, Rappuoli found that H. pylori puoli, ‘‘in large part because regulatory The development of the pertussis first attached to the eukaryotic cell and agencies and companies did not want to vaccine was particularly satisfying for Rap- then injected toxin directly through a change something that had been working puoli. In 1993, Italy adopted the use of hole in the membrane. ‘‘It was a totally for a century.’’ Thus, CRM197 was not the new vaccine, and within 2 years the new mechanism,’’ he says (6). Chiron, pursued as a diphtheria vaccine. disease was essentially eliminated. ‘‘So which was acquired by the pharmaceuti- However, CRM197 found an even that is basically one of the most beautiful cal company Novartis, has continued to broader application than for diphtheria. things that can happen if you do my probe the basic biology of the microbe. During the 1980s, other vaccine makers job....That’s been my motivation ever ‘‘We are starting to understand how were developing conjugate vaccines for since,’’ Rappuoli says. bacteria can cause cancer at the molecu- Hemophilus influenzae. Research by lar level, but we don’t have the full story John B. Robbins had established that Eliminating Serotype C Meningococcus yet,’’ says Rappuoli. The H. pylori toxin linking a polysaccharide to a carrier pro- ‘‘The next [vaccine] I thought I could triggers molecular changes in the cell tein could transform it into a powerful tackle with my team was meningococcus,’’ that resemble early stages of cancer. vaccine. Robbins suggested to Rappuoli says Rappuoli. Five primary serotypes ex- The work has led to many productive that CRM197 might make a good car- ist for Neisseria meningitides, the bacteria partnerships, including one with long- rier. ‘‘You vaccinate an infant with a responsible for meningitis and septicemia: time collaborator and fellow National polysaccharide, and there is absolutely A, B, C, Y, and W135. Rappuoli knew Academy of Sciences member Stanley no response. You link the polysaccha- from Gotschlich that capsule polysaccha- Falkow, a microbiologist at Stanford ride to a protein [such as CRM197], and rides were protective against serotype C. University (Stanford, CA) (7, 8). the response is dramatic. It’s the basis of That knowledge had been used to pro- As Rappuoli studied bacterial patho- many vaccines to date,’’ explains Rap- duce the meningococcal C vaccine, but genesis, he became increasingly aware puoli. Today, Rappuoli estimates that that microbiologists were growing bacte- every child in the United States and Eu- ria under artificial conditions that barely rope probably receives four or five vac- Eradicating whooping mimicked the real situations when these cinations with CRM197. It is a carrier microbes encountered humans. ‘‘We re- molecule for vaccines such as H. influen- cough in Italy was “one alized that some very important viru- zae, pneumococcus, and meningococcus. lence factors are not expressed when of the most beautiful you study the bacteria under those New Generation of Vaccines conditions,’’ says Rappuoli. He and his As with diphtheria, the new generation of things that can happen colleagues coined the term ‘‘cellular mi- vaccines against pertussis (whooping crobiology’’ to signal to the scientific cough) was made from a toxin that had if you do my job.” community, mainly to cell biologists and been deactivated with formaldehyde. ‘‘I microbiologists, that these two disci- didn’t waste any time doing that. I cloned plines should fuse into one. The idea, and sequenced the gene for pertussis the vaccine was not effective in infants. In which was published in a short essay in toxin and did what Pappenheimer had 1989, encouraged by promising results of Science, was immediately popular (9). A done 15 years before with diphtheria,’’ the first conjugate of CRM197 with H. new journal called says Rappuoli. But this time he and col- influenzae, Rappuoli and colleague Paolo was launched, two textbooks adopted league Mariagrazia Pizza used site- Costantino began making conjugate vac- the name, and several annual meetings directed mutagenesis to specifically alter cines for subtypes A and C. are now devoted to this new fusion field. amino acids in the active site of the toxin. Subtype C was mostly prevalent in the The result was a nontoxic molecule that United States, the United Kingdom, and Reverse Vaccinology made a potent vaccine (4). Europe, whereas subtype A was preva- While Rappuoli was working on meningo- The vaccine brought lent in Africa. Vaccines against both coccal vaccines, serotype B, which causes attention to Rappuoli and helped establish subtypes were effective in Phase I and II approximately 50% of meningitis cases Sclavo Research Center as a worldwide studies in the United Kingdom. Between worldwide, posed a greater challenge than leader in vaccine development. The center 1994 and 2000, Chiron worked with the the other subtypes did. Subtypes A, C, Y, became a part of the California-based government to develop and license the and W135 all have surface polysaccharides biotechnology company Chiron (Em- meningococcal C vaccine. In 1999, me- that can be coupled to proteins to yield eryville, CA). The pertussis vaccine also ningococcal C vaccinations began in the potent conjugate vaccines, but the only established a new generation of so-called United Kingdom, and within a year ev- polysaccharide on the capsule of subtype acellular vaccines, which, unlike older vac- ery person from 2 months to 18 years of B is polysialic acid, which is identical to cines, did not contain cells or cell age received a vaccination. By 2001, the the sugar on human cells. ‘‘So even by the fragments. disease was essentially eradicated. mid-1990s . . . there was no way to make a A National Institutes of Health (NIH; vaccine,’’ says Rappuoli of serotype B Bethesda, MD) trial revealed that Rap- H. pylori and Cellular Microbiology meningitis. puoli’s acellular vaccine was as effective as In the 1990s, Rappuoli began working In 1995, Craig Venter, head of The traditionally made vaccines, but with an on H. pylori, which was discovered in Institute for Genomic Research (TIGR; additional advantage—it required 10-fold the early 1990s and linked to gastric ul- Rockville, MD), sequenced the H. influ- fewer molecules (5). In 1995, this finding cers and cancers. H. pylori produced a enzae genome, and Rappuoli saw a new led regulatory agencies in the United toxin, but nobody had isolated it. He way to tackle subtype B. He visited

10832 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0604892103 Trivedi Downloaded by guest on September 28, 2021 Venter in 1997 and asked whether tion (CDC; Atlanta, GA), no vaccine with the vaccine and MF59 adjuvant TIGR would sequence N. meningitides was available. could recognize both the 1997 and subtype B. ‘‘When I explained the medi- In 2000, Rappuoli met with represen- 2003͞2004 avian flu virus strains (13). cal need and the opportunity to use a tatives from the New Zealand Ministry ‘‘So the adjuvant probably allows a new emerging technology to solve a very of Health. ‘‘I told them the problem was good immune response with a low anti- old problem, [Venter] was convinced it economic not technical,’’ he says. New gen dose but also helps to provide pro- would be worthwhile,’’ says Rappuoli. Zealand has a relatively small popula- tection against a virus that we know Within 18 months, the entire genome tion of 4 million people and was thus changes every year. So this shows two sequence of serotype B was in hand (10). not a lucrative market for vaccine com- reasons why there is no way to develop Fifty years of work on subtype B had panies. ‘‘I told them the New Zealand a vaccine against avian influenza with- yielded only about a dozen surface- government had to make the project out a good adjuvant,’’ says Rappuoli. exposed proteins as potential vaccine tar- attractive,’’ Rappuoli says. The govern- ‘‘That [finding] provides the opportu- gets; the completed genome sequence ment subsequently offered $200 million nity to really change our strategy by yielded more than 90. ‘‘It became immedi- to encourage vaccine development. Chi- which we tackle influenza pandemic.’’ ately clear that this was the new way for ron took up the challenge, and from Other than aluminum hydroxide and making vaccines, which I call reverse vac- 2004 to 2005, the entire population be- aluminum phosphate, MF59 is the only cinology, because we start with genes. tween 2 months and 18 years of age was other adjuvant approved for use in hu- This was the first time you didn’t need the vaccinated. ‘‘Now the disease is almost mans in the last century. Hundreds of pathogen and could go backwards from gone—it’s another powerful example of adjuvants have been tested in animals, but the information in the genome,’’ says how effective a vaccine can be if you do virtually all failed mostly because of toxic- Rappuoli. it right,’’ says Rappuoli. ity. MF59 was originally developed for use In his PNAS Inaugural Article (1), with the annual flu vaccine to enhance Rappuoli reveals the spoils of the ge- Avian Influenza and Adjuvants immune response in elderly people. nome sequence of subtype B meningo- In the last decade, another more insid- ‘‘When we were immunizing old mice, this coccus. He and his colleagues identified ious disease and its microbial source adjuvant allowed the old mice to respond five antigens that, when combined with has occupied Rappuoli’s attention: to the vaccination like the young mice. In an aluminum hydroxide adjuvant, confer avian influenza and the H5N1 virus. In fact, I call this adjuvant the ‘Viagra’ of the immunity in 78% of mice when chal- 1997, when avian flu arrived in Hong immune system.’’ (14) lenged with 85 strains of subtype B from Kong, Rappuoli’s team was the first to Rappuoli also is supervising the de- around the world. ‘‘I hope I’m right that make a vaccine against H5N1. In 1999, velopment of another adjuvant, called this vaccine will [eliminate] meningococ- Chiron launched a clinical trial in the LTK63. Derived from an Escherichia cus B globally,’’ says Rappuoli. Novartis United Kingdom to test the vaccine. coli toxin, LTK63 improves the immu- recently completed Phase I clinical trials Rappuoli’s team tested the vaccine nogenicity of mucosal vaccines. He for this serotype B vaccine. with and without a new adjuvant called speculates that engineering ‘‘immuno- MF59. Rappuoli says that much of genicity molecules,’’ such as this adju- Vaccinating New Zealand what is known about avian flu vaccines vant, will be a rapidly evolving field. One particularly significant achieve- was written in their paper published in ‘‘Once I complete the development of ment for Rappuoli was making a sero- The Lancet in 2001 (11). ‘‘But in 2001, vaccines for avian influenza and sero- group B meningococcus, I’ll feel pretty type B meningococcal vaccine specific nobody could care less about H5N1, satisfied,’’ says Rappuoli, ‘‘but I also for the people of New Zealand. Begin- and the paper went unnoticed,’’ he want to see reverse vaccinology applied ning in 1990, New Zealand became says. The paper contained two key to parasites and worms, which pose a mired in a decade-long epidemic of messages. The vaccine was ineffective huge medical burden.’’ He also suggests serotype B meningitis. The New Zea- without the adjuvant, but, with the ad- that using vaccines to treat chronic dis- land epidemic, unlike those in the juvant, only half the dose of antigen eases and cancer will become increas- United Kingdom and the United was required, essentially extending the ingly feasible as the understanding of States, was caused by a single bacterial vaccine’s manufacturing capacity. This innate immunity improves. Says Rap- strain. Even in the 1990s, the technical information is consistent with a recent puoli, ‘‘We are about to enter a new era solution for a single-strain vaccine was NIH paper, says Rappuoli (12). in immunotherapy—what I cannot tell is well known. Yet after a decade of dis- The MF59 adjuvant, which has been if it is 2 years away or 10 years away, ease and consultation with the World used since 1997, also has another ad- but it is going to be very exciting.’’ Health Organization and the U.S. Cen- vantage. Trials carried out by the CDC ters for Disease Control and Preven- showed that only people vaccinated Bijal Trivedi, Freelance Science Writer

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