Vaccines are one of the most powerful means for preventing epidemiological diseases. However, despite the high number of vaccines there are still many diseases that strike indiscriminately old and new generations, often carrying much more serious side effects causing great inconvenience and high costs.
Nowadays Pneumonia infections still represent one of the major causes of worldwide death, and in particular Streptococcus pneumoniae and its capsular polysaccharides are classified as one of the worst enemy, and the development of effective vaccines against these pathogens is one of the first aims of the World Health Organization.
This PhD work is focused on the synthesis of zwitterionic oligosaccharide analogues of the smallest immunogenic structure of the capsular polysaccharide repeating unit of Streptococcus pneumoniae type 14, as hypothetical proposal for the development of new vaccines.
SUPERVISOR
Prof. Giorgio Catelani
School of graduate
Studies
“Fisiopatologia clinica e
scienze del farmaco,
programma di scienze del
farmaco e delle sostanze
bioattive”
XXVI course
PhD Thesis
2011-2013
UNIVERSITY OF PISA
CHIM/06
DIRECTOR OF THE
SCHOOL
Prof. Adriano Martinelli
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“A l'alta fantasia qui mancò possa; ma già volgeva il mio disio e 'l velle, sì come rota ch'igualmente è mossa, l'amor che move il sole e l'altre stelle.”
CHAPTER 1.1 ... 2
VACCINES, SUCCESSES AND PROSPECTIVE ... 2
1.1.1 VACCINES: BIOLOGICAL, SOCIAL AND CULTURAL FACTOR ... 2
1.1.2 VACCINES THROUGHOUT HISTORY ... 3
1.1.3 RESPIRATORY INFECTIONS REMAIN THE ONLY LEADING INFECTIOUS CAUSE OF DEATH ... 5
CHAPTER 1.2 ... 10
STREPTOCOCCUS PNEUMONIAE: A PUBLIC ENEMY ... 10
1.2.1 STREPTOCOCCUS PNEUMONIAE ... 10
1.2.2 MULTI-DRUG-RESISTANCE (MDR) ... 13
1.2.3 VACCINES AGAINST S.PNEUMONIAE ... 15
CHAPTER 1.3 ... 21
ZWITTERIONIC POLYSACCHARIDES ... 21
1.3.1 ZWITTERIONIC POLYSACCHARIDES: UNCOMMON CAPSULAR POLYSACCHARIDES ... 21
1.3.1.1 Structural analysis ... 23
1.3.1.2 Molecular size effects on Zwitterionic immunologic activity ... 26
1.3.2 ZWITTERIONIC POLYSACCHARIDES AND IMMUNE SYSTEM ... 27
1.3.2.1 T-cell activation by ZPSs – adaptive immunomodulation ... 27
1.3.2.2 T-cell activation by ZPSs – innate immunomodulation ... 29
CHAPTER 1.4 ... 33
AIMS OF WORK ... 33
1.4.1 TARGETS AND THESIS AIMS ... 34
CHAPTER 2.1 ... 39
CHEMICAL RESULTS AND DISCUTION ... 39
2.1.1SYNTHESES OF 6-OH-Β-D-LACTOSAMINE GLYCOSYL ACCEPTORS 20 A-E ... 40
5.1.1.1 Amino sugar derivatives via N-benzyl intermediates ... 44
2.1.2 SYNTHESES OF LACTOSIDE TRICHLOROACETIMIDATE DONORS 21A-C ... 46
2.1.3 FINAL COUPLINGS GIVING TETRASACCHARIDES 22-28 AND DEPROTECTION STEPS ... 49
2.1.4 FURTHER DEVELOPMENTS ... 57
CHAPTER 2.2 ... 60
CONFORMATIONAL ANALYSIS ... 60
2.2.1MODELLING STUDIES ... 60
2.2.2NMR ANALYSES FOR CONFIRMING CONFORMATIONAL STUDIES ... 62
CHAPTER 2.3 ... 64
BIOLOGICAL TESTS ... 64
2.3.1BIOLOGICAL RESULTS AND DISCUSSION ... 64
2.3.2MATERIALS AND METHODS ... 66
IMMUNOGENIC SPECIFICITY TEST ... 67
GENERAL METHODS ... 68
3.1 SYNTHESES OF 6-OH-Β-D-LACTOSAMINE GLYCOSYL ACCEPTORS 20 A-E ... 69
Methyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyde (31a) ... 69
3-O-tosylpropyl 3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-β-D-glucopyranosyde (35) ... 69
3-azidopropyl 3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-β-D-glucopyranosyde (31b) ... 70
Methyl 2-acetamido-3-O-benzoyl-2-deoxy-4,6-O-isopropylidene-β-D-glucopyranosyde (32a) ... 70
3-azidopropyl 2-acetamido-2-deoxy-4,6-O-isopropylidene-β-D-glucopyranosyde (32b) ... 71
Methyl 2-acetoamido-3-O-benzoyl- 2-deoxy-4,6-O-isopropylidene β-D-glucopyranosyde (33a) ... 72
Methyl 2-acetamido-3-O-benzyl-2-deoxy-4,6-O-isopropylidene-β-D-glucopyranosyde (33b) ... 72
3-Azidopropyl 2-acetamido-3-O-benzoyl-2-deoxy-4,6-O-isopropylidene-β-D-glucopyranosyde (33c) ... 73
3-Azidopropyl 2-acetamido-3-O-benzyl-2-deoxy-4,6-O-isopropylidene-β-D-glucopyranosyde (33d) ... 73
General procedure for 4,6-O-isopropylidene removal ... 74
Methyl 2-acetamido-3-O-benzoyl-2-deoxy-β-D-glucopyranosyde (36a) ... 74
Methyl 2-acetamido-3-O-benzyl-2-deoxy-β-D-glucopyranosyde (36b) ... 74
3-Azidopropyl 2-acetamido-3-O-benzoyl-2-deoxy-β-D-glucopyranosyde (36c) ... 75
3-Azidopropyl 2-acetamido-3-O-benzyl-2-deoxy-β-D-glucopyranosyde (36d) ... 75
General procedure for 6-O selective protection with tert-butyldimethylsilyl chloride (TBDMSCl) ... 75
Methyl 2-acetamido-3-O-benzoyl-6-O-tert-butyldimethylsilyl-2-deoxy-β-D-glucopyranosyde (19a) ... 76
Methyl 2-acetamido-3-O-benzyl-6-O-tert-butyldimethylsilyl-2-deoxy-β-D-glucopyranosyde (19b) ... 76
3-Azidopropyl 2-acetamido-3-O-benzoyl-6-O-tert-butyldimethylsilyl-2-deoxy-β-D-glucopyranosyde(19c) ... 76
3-Azidopropyl 2-acetamido-3-O-benzyl-6-O-tert-butyldimethylsilyl-2-deoxy-β-D-glucopyranosyde (19d)... 77
General procedure for 4-O glycosylation ... 77
Methyl 4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-2-acetamido-3-O-benzoyl-6-O-t-butyldimethylsilyl-2-deoxy-β-D-glucopyranosyde (41a) ... 78 Methyl 4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-2-acetamido-3-O-benzyl-6-O-t-butyldimethylsilyl-2-deoxy-β-D-glucopyranosyde (41b) ... 78 3-Azidopropyl 4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-2-acetamido-3-O-benzoyl-6-O-t-butyldimethylsilyl-2-deoxy-β-D-glucopyranosyde (41c) ... 79 3-Azidopropyl 4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-2-acetamido-3-O-benzyl-6-O-t-butyldimethylsilyl-2-deoxy-β-D-glucopyranosyde (41d) ... 79
General procedure for 6-O selective deprotection... 80
Methyl 4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-2-acetamido-3-O-benzoyl-2-deoxy-β-D-glucopyranosyde (20a) ... 80 Methyl 4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-2-acetamido-3-O-benzyl-2-deoxy-β-D-glucopyranosyde (20b) ... 81 3-Azidopropyl 4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-2-acetamido-3-O-benzoyl-2-deoxy-β-D-glucopyranosyde (20c) ... 81 3-Azidopropyl 4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-2-acetamido-3-O-benzyl-2-deoxy-β-D-glucopyranosyde (20d) ... 82 Methyl 4-O-(2,3,4-tetra-O-acetyl-6-azido-6-deoxy-β-D-galactopyranosyl)-2-acetammido-3-O-benzyl-2-deoxy-β-D-glucopyranosyde (20e) ... 82
3.2 AMINO SUGAR DERIVATIVES VIA N-BENZYL INTERMEDIATES ... 84
4-O-(2,6-Di-O-methyl-3,4-O-isopropylidene-β-D-galactopyranosyl)-2,3:4,6-di-O-isopropylidene-aldehydo-D -glucose dimethyl acetal (43). ... 84 4-O-(2,6-Di-O-methyl-β-D-galactopyranosyl)-2,3-O-isopropylidene-aldehydo-D-glucose dimethyl acetal (44).84
4-O-(2,6-Di-O-methyl-3,4-O-isopropylidene-β-D
-galactopyranosyl)-2,3-O-isopropylidene-6-O-tosyl-aldehydo-D-glucose dimethyl acetal (42d). ... 85
3-O-Tosylpropyl 2-acetamido-2-deoxy-4,6-O-isopropylidene-β-D-glucopyranosyde (42e) ... 86
3-N-Benzylamminopropyl 2-acetamido-2-deoxy-4,6-O-isopropylidene-β-D-glucopyranosyde (46e) ... 87
General procedure for synthesis of N-benzyl intermediates (46 a-d) ... 87
4-O-(6-Benzylamino-6-deoxy-3,4-O-isopropylidene-β-D -galactopyranosyl)-2,3:5,6-di-O-isopropylidene-aldehydo-D-glucose dimethyl acetal (46a). ... 88
6-Benzylamino-6-deoxy-1,2:3,4-di-O-isopropylidene-α-D-galactopyranose (46b). ... 88
6-Benzylamino-6-deoxy-1,2:3,5-di-O-isopropylidene-α-D-glucofuranose (46c). ... 89
4-O-(2,6-Di-O-methyl-3,4-O-isopropylidene-β-D -galactopyranosyl)-6-benzylamino-6-deoxy-2,3-O-isopropylidene-aldehydo-D-glucose dimethyl acetal (46d). ... 89
General procedure for preparation of amino sugar derivatives (47a-d) by hydrogenolysis. ... 90
4-O-(6-Amino-6-deoxy-3,4-O-isopropylidene-β-D-galactopyranosyl)-2,3:5,6-di-O-isopropylidene-aldehydo-D -glucose dimethyl acetal (47a). ... 90
6-Amino-6-deoxy-1,2:3,4-di-O-isopropylidene-α-D-galactopyranose (47b). ... 90
6-Amino-6-deoxy-1,2:3,5-di-O-isopropylidene-α-D-glucofuranose (47c). ... 90
4-O-(2,6-Di-O-methyl-3,4-O-isopropylidene-β-D -galactopyranosyl)-6-amino-6-deoxy-2,3-O-isopropylidene-aldehydo-D-glucose dimethyl acetal (47d). ... 91
3.3 SYNTHESES OF LACTOSIDIC TRICHLOROACETIMIDATE DONORS 21A-C ... 92
4-O-[2-O-Benzyl-3,4-O-isopropylidene-β-d-galactopyranosyl uronate]-2,3:5,6-di-O-isopropylidene-aldeido-D-glucose dimethyl acetal (50b) ... 92
4-O-[6-O-Allyl-2-O-benzyl-3,4-O-isopropylidene-β-D-galactopyranosyl]-2,3:5,6-di-O-isopropylidene-aldeido-D -glucose dimethyl acetal (51a) ... 92
4-O-[6-O-Allyl-2-O-benzyl-β-d-galactopyranosyl]-2,3:5,6-di-O-isopropylidene-aldeido-D-glucose dimethyl acetal (52) ... 94
4-O-[6-O-Allyl-2,3,4-tri-O-benzyl-β-D-galactopyranosyl]-2,3:5,6-di-O-isopropylidene-aldeido-D-glucosie dimethyl acetal (50c) ... 94
General procedure for synthesis peracetyl derivatives 56b and c ... 95
4-O-[2-O-Benzyl-3,4-di-O-acetyl-β-D-galactopyranosyl uronate]-1,2,3,6-tetra-O-acetyl-D-glucopyranoside (56b) 95 4-O-[6-O-Allyl-2,3,4-tri-O-benzyl-β-D-galactopyranosyl]-1,2,3,6-tetra-O-acetyl-α,β-D-glucospyranosyde (56c)96 General procedure for synthesis of selective anomeric deprotection ... 96
4-O-[2-O-Benzyl-3,4-di-O-acetyl-β-D-galactopyranosyl uronate]-2,3,6-tri-O-acetyl-D-glucopyranose (57b) ... 97
4-O-[6-O-Allyl-2,3,4-tri-O-benzyl-β-D-galactopyranosyl]-2,3,6-tetra-O-acetyl-α,β-D-glucopyranose (57c) ... 97
General procedure for synthesis of selective anomeric deprotection ... 98
4-O-[2-O-benzyl-3,4-di-O-acetyl-β-D-galactopyranosyl uronate]-2,3,6-tri-O-acetyl-D-glucopyranosyl trichloroacetimidate (21b) ... 98
4-O-[6-O-Allyl-2,3,4-tri-O-benzyl-β-D-galactopyranosyl]-2,3,6-tetra-O-acetyl-α-D-glucopyranosyl trichloroacetimidate (21c) ... 98
3.4 FINAL COUPLINGS GIVING TETRASACCHARIDES 22-28 AND DEPROTECTION STEPS ... 100
General procedure for 6-O glycosylation ... 100
Methyl 2-acetammido-3-O-benzoyl-4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-6-O-[4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-2,3,6-tri-O-acetyl-β-D-glucopyranosyl]-2-desossi-β-D-glucopyranosyde (22) 100 3-Azidopropyl 2-acetammido-3-O-benzoyl-4-O-(2,3,4,6-tetra-O-acetyl-β-D -galactopyranosyl)-6-O-[4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-2,3,6-tri-O-acetyl-β-D-glucopyranosyl]-2-deoxy-β-D -glucopyranosyde (23) ... 101
Methyl 2-acetammido-3-O-benzyl-4-O-(2,3,4,6-tetra-O-acetyl-β-D -galactopyranosyl)-6-O-[4-O-(2-O-benzyl-3,4-di-O-acetyl-β-D-galactopyranosyl uronate)-2,3,6-tri-O-acetyl-β-D-glucopyranosyl]-2-deoxy-β-D
-glucopyranosyde (24) ... 101 3-Azidopropyl 2-acetammido-3-O-benzyl-4-O-(2,3,4,6-tetra-O-acetyl-β-D -galactopyranosyl)-6-O-[4-O-(3,4-di-O-acetyl-2-benzyl-β-D-galactopyranosyl uronate)-2,3,6-tri-O-acetyl-β-D-glucopyranosyl]-2-deoxy-β-D -glucopyranosyde (25) ... 102 Methyl 2-acetammido-3-O-benzyl-4-O-(2,3,4-tri-O-acetyl-6-azido-6-deoxy-β-D -galactopyranosyl)-6-O-[4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-2,3,6-tri-O-acetyl-β-D
-glucopyranosyl]-2-deoxy-β-D-glucopyranosyde (26) ... 103 Methyl 2-acetammido-3-O-benzyl-4-O-(6-azido-2,3,4-tri-O-acetyl-6-deoxy-β-D -galactopyranosyl)-6-O-[4-O-(3,4-di-O-acetyl-2-benzyl-β-D-galactopyranosyl uronate)-2,3,6-tri-O-acetyl-β-D-glucopyranosyl]-2-deoxy-β-D -glucopyranosyde (27) ... 104 Methyl 2-acetammido-3-O-benzyl-4-O-(2,3,4,6-tetra-O-acetyl-β-D -galactopyranosyl)-6-O-[4-O-(6-O-allyl-2,3,4-tri-O-acetyl-β-D-galactopyranosyl)-2,3,6-tri-O-acetyl-β-D-glucopyranosyl]-2-deoxy-β-D
-glucopyranosyde (28) ... 104 Methyl 2-acetammido-4-O-(β-D-galactopyranosyl)-6-O-[4-O-(β-D-galactopyranosyl)- β-D -glucopyranosyl]-2-deoxy-β-D-glucopyranosyde (5) ... 105 3-Azidopropyl 2-acetammido -4-O-β-D-galattopyranosyl-6-O-[4-O-(β-D-galattopyranosyl)-β-D
-glucopyranosyl]-2-deoxy-β-D-glucopyranosyde (58) ... 106 3-Propylammonium chloride 2-acetammido -4-O-β-D-galattopyranosyl-6-O-[4-O-(β-D-galattopyranosyl)-β-D -glucopyranosyl]-2-deoxy-β-D-glucopyranosyde (7) ... 106 Methyl 2-acetammido-4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-6-O-[4-O-(3,4-di-O-acetyl-β-D -galactopyranosyl uronyl)-2,3,6-tri-O-acetyl-β-D-glucopyranosyl]-2-deoxy-β-D-glucopyranosyde (63) ... 107 Methyl 2-acetammido-4-O-(β-D-galactopyranosyl)-6-O-[4-O-(β-D-galactopyranosyl uronyl)- β-D
-glucopyranosyl]-2-deoxy-β-D-glucopyranosyde (6) ... 108 3-Amminopropyl 2-acetammido-4-O-(2,3,4,6-tetra-O-acetyl-β-D -galactopyranosyl)-6-O-[4-O-(3,4-di-O-acetyl-2-benzyl-β-D-galacturonic acid)-2,3,6-tri-O-acetyl-β-D-glucopyranosyl]-2-deoxy-β-D-glucopyranosyde
(64) 108
3-Amminopropyl 2-acetammido-4-O-β-D-galattopyranosyl-6-O-[4-O-(β-D-galacturonic acid)-β-D
-glucopyranosyl]-2-deoxy-β-D-glucopyranosyde (2) ... 109 Methyl 2-acetammido-4-O-(6-ammino-2,3,4-tri-O-acetyl-6-deoxy-β-D -galactopyranosyl)-6-O-[4-O-(3,4-di-O-acetyl-β-D-galacturonic acid)-2,3,6-tri-O-acetyl-β-D-glucopyranosyl]-2-deoxy-β-D-glucopyranosyde (65) .... 110 Methyl 2-acetammido-4-O-β-D-galactopyranosyl-6-O-[4-O-(β-D-galacturonic acid)-β-D -glucopyranosyl]-2-deoxy-β-D-glucopyranosyde (3) ... 110 Methyl 2-acetammido-3-O-benzyl-4-O-(6-azido-6-deoxy-β-D-galactopyranosyl)-6-O-[4-O-(β-D
-galactopyranosyl)-β-D-glucopyranosyl]-2-deoxy-β-D-glucopyranosyde (66) ... 111 Methyl 2-acetammido-4-O-(6-amino-6-deoxy-β-D-galactopyranosyl)-6-O-[4-O-(β-D-galactopyranosyl)-β-D -glucopyranosyl]-2-deoxy-β-D-glucopyranosyde chloride (8) ... 111 Methyl 2-acetammido-3-O-benzyl-4-O-(2,3,4,6-tetra-O-acetiyl-β-D -galactopyranosyl)-6-O-[4-O-(2,3,4-tri-O-acetyl-β-D-galactopyranosyl)-2,3,6-tri-O-acetyl-β-D-glucopyranosyl]-2-deoxy-β-D-glucopyranosyde (67) .... 112 4-O-[6-O- Benzyl phosphorodiamidite-2-O-benzyl-3,4-O-isopropylidene-β-D -galactopyranosyl]-2,3:5,6-di-O-isopropylidene-aldeido-D-glucose dimethyl acetal (84) ... 113
1.
Introduction
Chapter 1.1
Vaccines, successes and prospective
1.1.1
Vaccines: biological, social and cultural factor
The history of vaccines and immunization began with the story of Edward Jenner, a country doctor from Berkeley (Gloucestershire), England, who in 1796 performed the world’s first vaccination.1
Janner observed the local customs of farming communities and the awareness that milkmaids, infected with cowpox (visible as pustules on their hand or forearm), were immune to subsequent outbreaks of smallpox that periodically swept through the area, led him to apply the scientific method of observation and experimentation.
He conduced one of the world’s first clinical trials; in fact he took pus from a cowpox lesion on a milkmaid’s hand and inoculated an eight-year-old boy, James Phipps. Six weeks later Jenner variolated two sites on Phipps’s arm with smallpox, yet the boy was unaffected by this as well as subsequent exposures.2 Based on twelve such experiments and sixteen additional case histories he had collected
since the 1770s, Jenner published at his own expense a volume that swiftly became a classic text in the annals of medicine: Inquiry into the Causes and Effects of the Variolae Vaccine. His assertion “that the cow-pox protects the human constitution from the infection of smallpox” laid the foundation for modern vaccinology.3
Although Jenner’s milkmaid experiments may now seem like quaint fables, they provided the scientific basis for vaccinology. Our current conceptions of vaccine development and therapy are now much more encompassing and firmly rooted in the science of immunology thank to the French chemist Louis Pasteur, who developed what he called a rabies vaccine in 1885, until this time in fact the word "vaccine" referred only to cowpox inoculation for smallpox.
Although what Pasteur actually produced was a rabies antitoxin that functioned as a post infection antidote, he expanded the term beyond its Latin association with cows and cowpox to include all inoculating agents.4 Thus, we largely have to thank Pasteur for today’s definition of vaccine as a
“suspension of live (usually attenuated) or inactivated microorganisms (e.g., bacteria or viruses) or fractions thereof administered to induce immunity and prevent infectious disease or its sequelae.”5
Starting from “Jennerian inoculation” discovery, governments and health organizations, which were founded, invested a lot on development and widespread distribution of safe, effective and affordable
vaccines, so that many of devastating diseases (measles, diphtheria, smallpox, pertussis, etc.) have been contained, especially in industrialized nations.
Ironically, as vaccines have become more commonplace, they have lost some of their allure, particularly to public funding agencies and public opinion. One might argue that vaccines have worked so well that many people now take them for granted. In this sense, scientific success has paradoxically contributed to the current problems with adequate funding mechanisms.6
Furthermore, vaccines throughout their history have always found social minds against their use; in fact, firstly they were considered as intrusion of privacy and bodily integrity, but with the passing of time they found adversaries due to religious belief and antivivisectionism. Moreover, nowadays the threat posed by false or unsubstantiated rumours about vaccines’ safety and the increasing in concern about the potential side-effects of vaccines, especially in industrialized countries, are also a barrier to progress, so that we can even speak of “antivaccinationism”. For these reasons vaccines security protocols are made, used, and tested in accordance with internationally accepted standards, this is one part of the effort to reduce the likelihood of a vaccine producing an adverse event. On the other part, an efficient post-marketing surveillance and investigation system have been instituted that will rapidly pick up and verify any rumours or reports of adverse events allegedly related to the use of a vaccine.7
In the end, the story is more complicated than it might appear at first glance. Even as existing vaccines continue to exert their immunological power and new vaccines offer similar hopes, remerging and newly emerging infectious diseases threaten the progress made. Furthermore, obstacles have long stood in the way of the production of safe and effective vaccines. The historical record shows that the development of vaccines has consistently involved sizable doses of ingenuity, political skill and irreproachable scientific methods. When one or more of these has been lacking or perceived to be lacking, vaccination has provided responses ranging from a revised experimental approach in the laboratory to a supply short age and even insurrection in the streets. In short, vaccines are powerful medical interventions that induce powerful biological, social, and cultural reactions.6
Nevertheless, if you asked a public health professional to draw up a top-ten list of the achievements of the past century, he or she would be hard pressed not to rank immunization first. Millions of lives have been saved and microbes stopped in their tracks before they could have a chance to wreak havoc. Briefly the vaccine represents the single greatest promise of biomedicine: disease prevention.
1.1.2
Vaccines throughout history
The 18th Century saw the growth in industrialization that brought more and more people to cities in search of work. While this led to overcrowding, poor sanitation and subsequent epidemics, there was a growing recognition of the nature of disease. There were two prevailing views of the causes of epidemics: Miasmic and Contagion. Both have public health implications.
Miasmic: this theory held that epidemics stemmed from certain atmospheric conditions and from miasmas rising from organic materials.
The growing morbidity and mortality developed during these years, especially among children has pushed biomedical science and public health to the greatest achievement of Vaccines.
The first method of vaccination, as we said, was invented by Edward Jenner (in 1796 against smallpox). The method involved taking material from a blister of someone infected with cowpox and inoculating it into another person’s skin; this was called arm-to-arm inoculation. However by the late 1940s, scientific knowledge had developed enough, so that large-scale vaccine production was possible and disease control efforts could begin in earnest.
The next routinely recommended vaccines were developed early in the 20th century. These included
vaccines that protect against pertussis (1914), diphtheria (1926), and tetanus (1938). These three vaccines were combined in 1948 and given as the DTP vaccine. In 1955, with the economical contribution of millions of Americans, was licensed the polio vaccine. In 1963 the measles vaccine was developed, and by the late 1960s, vaccines were also available to protect against mumps (1967) and rubella (1969).
Thus before 1980, thanks to the incoming technologies, introduction of essential hygienic rules and development of licensed and recommended vaccines for 8 of the most common diseases associated to other health inventions, millions of people were saved (Tab. 1.1),8 especially children giving them a
chance to grow up healthy, go to school and improve their life prospects. Table 1.1
Vaccine-Preventable
Disease Causes Deaths Causes Deaths
Vaccine Date(s), y Cases, 2006 Deaths 2004 Cases Deaths Diphteria 21 053 (1936-1945) 1822 (1936-1945) 30 508 (1938) 3065 (1936) 1928-1943 0 0 21053 (100) 1822 (100) Measles 530 217 (1953-1962) 440 (1953-1962) 763 094 (1958) 552 (1958) 1963, 1967, 1968 55 0 530 162 (99.9) 440 (100) Mumps 162 344 (1963-1968) 39 (1963-1968) 212 932 (1964) 50 (1964) 1940, 1967 6584 0 155 760 (95.9) 39 (100) Pertussis 200 752 (1934-1943) 4034 (1934-1943) 265 269 (1934) 7518 (1934) 1914-1941 15 631 27 185 120 (92.2) 4007 (99.3) Poliomyelitis, acute 19 794 (1941-1950) 1393 (1941-1950) 42 033 (1949) 2720 (1949) 1955, 1961-1963, 1983 0 0 19 794 (100) 1393 (100) Poliomyelitis, paralytic 16 316 (1951-1954) 1879 (1951-1954) 21 269 (1952) 3145 (1952) 1955, 1961-1963, 1987 0 0 16316 (100) 1879 (100) Rubella 47 754 (1966-1968) 17 (1966-1968) 488 796 (1964) 24 (1968) 1969 11 0 47734 (99.9) 17 (100) Congenital rubella Syndrome 152 (1966-1969) Not available 20 000 (1964-1965) 2160 (1964-1965) 1969 1 0 47 734 (99.9) 17 (100) Smallpox 29 005 (1900-1949) 337 (1900-1949) 110 672 (1920) 2510 (1902) 1798 0 0 29 005 (100) 337 (100) Tetanus 580 (1947-1949) 472 (1947-1949) 601 (1948) 511 (1947) 1933-1949 41 4 539 (92.9) 468 (99.2)
Historical Comparison of Morbidity and Mortality in US for vaccine.preventable deseases with vaccines licensed or recomanded before 1980: Diphteria, Measles, Mumps, Pertussis, Poliomyelitis, Rubella, Smallpox, Tetanus7
Estimated Annual Average
Prevaccine Estimated Annual No. Vs Most Recent
Reported No. (%Reduction) Most Recent Postvaccine Reported No. Prevaccine No. Peak
Vaccine-preventable diseases also constitute the major causes of illness and long-term disabilities among children both in industrialized and developing countries. These reasons added to the high costs of hospitalizations associated to these pathologies, have induced in the last twenty years the development of more vaccines than were produced in the past century providing and high reduction of
hospitalizations number (Tab. 1.2). [Hepatitis a (2000) and b (1985), meningococcal meningitis (2005), rotavirus diarrhoeal disease (1999), avia influenza caused by the H5N1 virus (2007), pneumococcal disease (1983, 2000, 2010), cervical cancer caused by human papillomavirus (2008)].8
Table 1.2
Vaccine-Preventable
Disease
Causes Hospitali-
zations Deaths Causes Deaths Vaccine Date(s), y Reported cases Estima-ted Cases Estima-ted Hospitali zations
Deaths Cases Hospitali- zations Deaths Hepatitis A 117 333 (1986-1995) 6863 (1986-1995) 137 (1986-1995) 254 518 (1971) 298 (1971) 1995 3579 15 298 895 18 102 035 (87.0) 5968 (87.0) 119 (86.9) Acute Hepatitis B 66 232 (1982-1991) 7348 (1982-1991) 237 (1982-1991) 74361 (1985) 267 (1985) 1981, 1986 4713 13 169 1461 47 53 063 (80.1) 5887 (80.1) 190 (80.2) Invasive Hemophilus influenzae type b 20 000 (1980s) Not available 1000 (1980s) Not available Not available 1985, 1987, 1990 208 (29 type b; 179 type unknown) <50 (2005) Not available <5 (2005) 19 950 (>99.8) Not available 995 (>99.5) PeInvasive Pneumococcal disease 63 067 (1997-1999) Not available 6500 (1997-1999) 64 400 (1999) 7300 (1999) 2000 5169 41 550 (2005) Not available 4850 (2005) 21 517 (34.1) Not available 1650 (25.4) Varicella 4 085 120 (1990-1994) 10 632 (1988-1995) 105 (1990-1994) 5 358 595 (1988) 138 (1973) 1995 48445 612 768 1276 19 (2004) 3 472 352 (85.0) 9356 (88.0) 86 (81.9)
Historical Comparison of Morbidity, Mortality and Hospitalizations in US for vaccine-preventable deseases with vaccines licensed or recomanded between 1980-2005.7
Prevaccine Estimated Annual No. Vs Most Recent Reported No. (%Reduction) Prevaccine No.
Most Recent Postvaccine Reported No. 2006
Estimated Annual Average Estimated Peak
These data demonstrate the wide success of vaccines and are the base of their dynamic development.
Another important challenge remains reaching people of all over the world, in fact following the Millennium Development Goal 4 (MDG 4), which calls for reducing the under-five mortality rate by two-thirds between 1990 and 2015, the world has made substantial progress, reducing the under-five mortality rate 47 percent (from 90 death per 1.000 live births in 1990 to 48 in 2012),9 but the rate of
this reduction in under-five mortality is still insufficient to reach the MDG 4 target. Furthermore iniquities in child mortality between high-incomes and low-incomes countries remain large. Historical trends show that progress for most countries has been too slow and that only 13 of the 61 countries with high under-five mortality rates are currently on track to achieve MDG 4. Many countries still have high under-five mortality rates, particularly those in Sub-Saharan Africa, home to all 16 countries with an under mortality rate above 100 deaths per 1.000 births.
1.1.3
Respiratory infections remain the only leading infectious
cause of death
In 2011 was estimated that 55 million people died worldwide.10 Ischemic heart disease (7 million),
stroke (6.2 million), lower respiratory infections (3.2 million), chronic obstructive lung disease (cancers, dementia, chronic obstructive lung disease or diabetes; 3 million), diarrhoea and HIV/AIDS (1.6 million) have remained the top major killers during the past decade. Non-Communicable Diseases (NCDs, the four main NCDs are: cardiovascular diseases, cancers, diabetes and chronic long diseases)
were responsible for two-thirds (36 million) of all deaths globally in 2011, up from 60% (31 million) in 2000. Cardiovascular diseases alone killed nearly 2 million more people in 2011 than in the year 2000. Tobacco use is a major cause of many of the world’s top killer diseases – including cardiovascular disease, chronic obstructive lung disease and lung cancer. In total, tobacco use is responsible for the death of about 1 in 10 adults worldwide. Smoking is often the hidden cause of the disease recorded as responsible for death.10
Figure 1.1
Tuberculosis, while no longer among the 10 leading causes of death in 2011, was still among the 15 causes, killing one million people in 2011. Maternal deaths have dropped from 420 000 in the year 2000 to 280 000 in 2011, but are still unacceptably high: nearly 800 women die due to complications of pregnancy and childbirth every day.10
Injuries continue to kill 5 million people each year. Road traffic injuries claimed nearly 3500 lives each day in 2011 – about 700 more than in the year 2000 – making it among the top 10 leading causes in 2011 (Fig.1.1).
In terms of number of deaths, 26 million (nearly 80%) of the 36 million of global NCD deaths in 2011 occurred in low- and middle-income countries. In terms of proportion of deaths that are due to NCDs, high-income countries have the highest proportion – 87% of all deaths were caused by NCDs – followed by upper-middle income countries (81%). The proportions are lower in low-income countries (36%) and lower-middle income countries (56%).10
In high-income countries, 7 in every 10 deaths are among people aged 70 years and older. People predominantly die of chronic diseases: cardiovascular diseases, cancers, dementia, chronic obstructive lung disease or diabetes. Lower respiratory infections remain the only leading infectious cause of death. Only 1 in every 100 deaths is among children under 15 years.10
In low-income countries, nearly 4 in every 10 deaths are among children under 15 years, and only 2 in every 10 deaths are among people aged 70 years and older; people predominantly die of infectious
diseases: lower respiratory infections, HIV/AIDS, diarrhoeal diseases, malaria and tuberculosis collectively account for almost one third of all deaths in these countries. Complications of childbirth due to prematurity, and birth asphyxia and birth trauma are among the leading causes of death, claiming the lives of many newborns and infants.10
In 2012, 6.6 million children died before reaching their fifth birthday; almost all (99%) of these deaths occurred in low- and middle-income countries.11 About 44% of deaths in children younger than
5 years in 2012 occurred within 28 days of birth – the neonatal period:preterm birth, intrapartum-related complications (birth asphyxia or lack of breathing at birth), and infections cause the most neonatal deaths.From the end of the neonatal period and through the first five years of life, the main causes of death are pneumonia, diarrhoea and malaria. Malnutrition is the underlying contributing factor in about 45% of all child deaths, making children more vulnerable to severe diseases (Fig. 1.2).11
Mortality from pneumonia generally decreases with age until late adulthood. Elderly individuals, however, are at particular risk for pneumonia and associated mortality. Because of the very high burden of disease in developing countries and because of a relatively low awareness of the disease in industrialized countries, the global health community has declared November 12 to be World Pneumonia Day, a day for concerned citizens and policy makers to take action against the disease.
Figure 1.2
Moreover Pneumonia is a major threat to older people, with an annual incidence for non-institutionalized patients estimated between 25 and 44 per 1000 population, up to four times that of patients younger than 65. Older residents of chronic care institutions have an incidence of 33 to 114 cases per 1000 population per year. It was stated that at any given moment as many as 2% of nursing-home residents may have pneumonia.12 Mortality rates for older patients in hospital-based studies of
community-acquired pneumonia (CAP) are reported to be as high as 30%. For nursing-home acquired pneumonia (NHAP), mortality rates may reach 57%. The diagnosis of pneumonia in this age group is often delayed because of the frequent absence of fever, the paucity or absence of cough, and changes in mental status (delirium), which further contributes to the high morbidity and mortality. Hospitalizations for CAP are also an indicator of adverse prognosis at 1 year in older patients: in a case-control study of 158 960 CAP patients versus 794 333 hospitalized controls; 1-year mortality was 41% for the CAP patients versus 29% for the control population.12
Pneumonia is caused by a various number of infectious agents, including viruses, bacteria and fungi, and the most common are:13
•Streptococcus pneumoniae – the most common cause of bacterial pneumonia in children and adults (over 65);
•Haemophilus influenzae type b (Hib) – the second most common cause of bacterial pneumonia;
•respiratory syncytial virus is the most common viral cause of pneumonia;
•in infants infected with HIV, Pneumocystis jiroveci is one of the commonest causes of pneumonia, responsible for at least one quarter of all pneumonia deaths in HIV-infected infants.
Thus development of several vaccines against these pathogens in addition to the use of other pneumonia-control measures (reduction of exposure to known risk factors, such as indoor pollutants, tobacco smoke, premature weaning and nutritional deficiencies) obviously represent the best prevention forms against epidemiological diffusion of pneumonia.
References
1 Baxby, D. Jenner’s Smallpox Vaccine: The Riddle of Vaccinia Virus and Its Origin (Heinemann Educational Books) 1981; Baxby, D. Smallpox Vaccine, Ahead of Its Time 2001; and Baxby, D. Vaccination: Jenner’s Legacy (Berkeley,
U.K.: Jenner Educational Trust, 1994).
2 Barquet N. and Domingo, P. Annals of Internal Medicine, 1997, 127, 8, 635–642. 3 Jenner, E. Inquiry into the Causes and Effects of the Variolae Vaccine 1798, 45. 4 Hansen, B. American Historical Review 1998, 103, 2 373–418.
5 Advisory Committee on Immunization Practices and the American Academy of Family Physicians, “General
Recommendations on Immunization,” Morbidity and Mortality Weekly Report 51”, 2002, RR02, 34.
6 Stern, A.M.; Markel, H. Health Affairs 2005, 24, 3, 611-621.
7 WHO report “State of the world’s vaccines and immunization” 2009, third edition. 8 Roush, S.W.; Murphy T.V.; JAMA 2007, 298, 18, 2155-2163.
9 UNICEF report “Levels & Trends in Child Mortality” 2013. 10 WHO report “The top 10 causes of death” 2009, third edition.
11 WHO report “Children: reducing mortality” 2012 September, Fact sheed 178. 12 Janseens, J.P.; Krause, K. H.; Lancet Infect. Desease 2004, 4, 112-124. 13 WHO report “Pneumonia” 2013 April, Fact sheed 331.
Streptococcus pneumoniae: a public enemy
1.2.1
Streptococcus pneumoniae
Streptococcus pneumoniae (S. pneumoniae or pneumococcus) was discovered more than 100 years ago and represents the major cause of illness and death worldwide.
S. pneumoniae is a gram positive bacterium in the shape of slightly pointed cocci. They are usually found in pairs (diplococci), but are also found singly and in short chains. S. pneumoniae are alpha hemolytic (a term describing how the cultured bacteria break down red blood cells for the purpose of classification). S. pneumoniae are found normally in the upper respiratory tract, including the throat and nasal passages and is characterized by a polysaccharide capsule that completely encloses the cell (Fig. 1.3).
Figure 1.3
The primary S. pneumoniae virulence factor is represented by polysaccharides constituting capsules, provided with anti-phagocytosis power, which added to toxic substances producing (pneumolysin and autolysin), increasing diffusion and invasiveness of bacteria. The polysaccharide capsule is also capable to neutralize specific antibodies produced during advanced infection phase.1 S.
pneumoniae is a commensal of the human nasopharynx bacteric flora and primarily transmitted from person to person via contact with respiratory droplets.2 Transmission typically occurs when a carrier
(normally a healthy person which has the potential to be a source of infection for others) coughs or sneezes within six feet of other people, potentially infecting them.
This class of bacteria is able to migrate to other niches within the human body to cause various diseases of the upper respiratory tract such as pneumonia, otitis media, sinusitis and bronchitis;
furthermore if the immune system is not able to validly counteract the pathogen can get to generate mastoiditis and meningitis or, in the end, to reach the thoracic duct, entering the bloodstream causing bacteremia, sepsis and other conditions such as bone and joint infection and peritonitis.
Nasopharyngeal carriage of S. pneumoniae is more common in young children than adults and varies by geographic region. Variation in the prevalence of nasopharyngeal carriage may be due to socioeconomic conditions such as crowding, sanitation, family size, and day care contact, and to genetic differences in the host that affect the likelihood of nasopharyngeal colonization. The duration of carriage also varies, depending on the host’s age and on the serotype of the colonizing strain, and typically ranges between 1 and 17 months.
Humoral defence factors as antibodies complement and phagocytic cells are mainly opponents against S. pneumoniae, therefore deficits of antibody formation have the greatest impact on pneumococcal infections.
For this reason more commonly affected social classes by S. Pneumoniae are individuals who are infected with HIV or who have underlying ematic conditions or neutropenia, such as sicklecell disease, children aged <5 years and adults older 65 years.3 (Fig. 1.4)
Figure 1.4
S. pneumoniae is the first cause of childhood diseases in developing-country, with more 500/100.000 infected among children aged <1 year, and mortality amounting 50% in case of Invasive Pneumococcal Diseases (IPDs).4 Every year more than one million on 2.6 million of deaths among
children <5 years old, caused by acute respiratory infections (Fig. 1.5), are attributed to S. pneumoniae. Figure 1.5
The higher incidence is in developing-countries, in particularly in India (Fig. 1.5), and substantially higher incidence rates were reported in blacks than in whites, and even children under 6 months living in Latin America appear to be at greater risk as well as those Native Americans, aboriginal Australian population and people from some minorities Israeli.4
In Europe was estimated rate of IPD 462/100.000 children <5 year, counting 29/100.000 deaths per year, instead adults (>65 years) counted 223/100.000 infections per year, 6, 5 of which lead to death.5 In United States 40.000 cases of invasive pneumococcal disease (IPD), resulting in 4.000
deaths still occurred among adults in 20106 and was demonstrated that the incidence among people
with comorbid conditions such as diabetes, cancer, chronic pulmonary disease, renal disease, HIV/AIDS, liver disease and alcohol abuse, remains alarming. Finally among adults 1.6-10.6 cases on 1.000 people of pneumococcal pneumonia are counted, with a mortality rate of 20-30% also in industrialized countries with major death incidence among high-risk adults.
On the basis of capsules chemical structure 94 immunologically distinct serotypes of S. pneumoniae have been described including recently identified serotypes 6C, 6D, 20A/20B and 11E, and each produces a biochemically distinct polysaccharidecapsule that is in most cases covalently attached to the cellwall. Serotype affects nearly every aspect of pneumococcalpathogenesis and of nasopharyngeal carriage, which precedesdisease and serves as the reservoir for transmission of the organism. The most common serotypes in both invasive disease and carriage show remarkable consistency across geography and time despite some differences in the details. Serotypes differ not only in their prevalence, but also in their tendency to causeinvasive or mucosal disease (ratio of disease cases to carriers),their age distribution, their tendency to cause outbreaks, and their degree of antimicrobial resistance.
Recent Meta-Analysis7 of serotype-specific diseases, among patients with pneumonia and
meningitis, suggest that outcome IPDs, like other epidemiologic measures, are stable serotype associated properties; in fact the rank orders of serotypes found in nasopharyngeal carriage and invasive disease are similar worldwide, with a few exceptions. Serotypes 1, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 12F, 14, 19A, 19F, 22F, 23F were ranked as major causes of mortality by pneumonia, whereas serotypes 1, 6A, 6B, 7F, 8, 9N, 9V, 12F, 14, 19F, 23 were classified as major causes of meningitis.
In particularly in the US most IPDs were associated with serotypes: 6A, 6B, 9V, 14, 19A, 19F, and 23 F; instead according studies of Imöhl,8 conducted in Germany with the involvement of the German
National Reference Centre for Streptococci and the collection of data 1992-2008, allows to identify the serotype 14 as the most commonly involved in the pathogenesis of invasive infections, followed by serotypes 3, 7F, 1 and 23F. The serotype 14 was proved the most common in Ireland in the period both before and after the introduction of pneumococcal vaccination.9 A similar study conducted in Denmark
from 1938 to 2007, with the involvement of the national laboratories surveillance,10 has highlighted
the emergence of infections caused by serotypes 4 and 19A and serogroup 9.
The infections caused by serotype 14 seem to have declined with the introduction of vaccination against pneumococcal disease, but it is always one of the most common serogroup worldwide and it’s also used as the reference,because it is a common cause of IPDs and is the only serotype that contains
nonzero numbers of fatalities in the most studies.7 The 2011 annual report of the European
Antimicrobial Resistance Surveillance Network (EARS-Net) has characterized the serotypes of S. pneumoniae, analyzed by the reference laboratories, showing that the most incoming-common serotypes are: 1 (14% ), 19 (13%) , 7 ( 12%), 3 (9 %). The serotype 1 is therefore one of the most important and emerging serotypes, as well as the 19A and 7F (Fig. 1.6).
Figure 1.6
1.2.2
Multi-drug-resistance (MDR)
Over the past 3 decades, antimicrobial resistance among Streptococcus pneumoniae, the most common cause of community-acquired pneumonia (CAP), has escalated dramatically worldwide. In the late 1970s, strains of pneumococci displaying resistance to penicillin were described in South Africa, Australia and Spain. By the early 1990s, penicillin-resistant clones of S. pneumoniae spread rapidly across Europe and globally with a certain geographic variability since the phenomenon of diffusion is more common in South Africa and the Far East. A survey of 15 European countries (2004–05) noted striking differences in total antibiotic use and rates of resistance among different countries.11 Rates of
penicillin resistance ranged from 0% (Denmark) to 57.1% (Greece) instead macrolide resistance ranged from 6.9% (Norway) to 57.1% (Greece). (Fig. 1.7)
Figure 1.7
Europe Penicillin resistance Europe Macrolides resistance (Erythromycin)
The 2011 report, produced by European Antimicrobial Resistance Surveillance System (EARSS- Net), describes the trend of penicillin resistance of S. pneumoniae bringing the increase of the strains not susceptible from 10.3 % in 2004 to 23.1% in 2008 with a stabilization of around 20% in the years ( 20.2% in 2009, 18.2% in 2010 , 19.6% in 2011).12 In Europe the percentage of not-sensitive
pneumococcal serotypes to penicillin was 8.8% with a growing trend in Denmark, Estonia, Lithuania and Sweden, and a reduction in Belgium, Portugal, Hungary and France.13 Conversely, the absence of
susceptibility to macrolides seems to be more significant with a value of 14.6% in 2011, an increasing trend has been observed in Lithuania, Slovenia and Spain while one decrease was found in Hungary, Norway and Portugal.13
The classes of antibiotics that are of particular attention are the β-lactam (penicillins, cephalosporins, and carbapenems), macrolides (erythromycin, azithromycin, clarithromycin and lincosamides), tetracycline, inhibitors of folate (trimethoprim, trimoxazole), fluoroquinolones (ciprofloxacin, levofloxacin, gemifloxacin, moxifloxacin).
The spread of the phenomenon is due essentially to the expansion of some clones; in particular penicillin- and macrolide-resistant isolates are derived from five serotypes: 6B, 9V, 14, 19F, 23F.14 The
evidence, however, point out the important increase of resistant strains penicillin belonging to serotype 19A.15 The reason why these particular serotypes present higher probability of containing
antibiotic resistance determinants is not yet clear, one hypothesis is that these serotypes are common among children which may be carrier for longer duration and thus expose these stains to increased antibiotic pressure.16
There are several risk factors for the acquisition of antibiotic-resistant strains (genetic differences in the host, socioeconomic conditions, access to health care centres daily for children, recent infections, respiratory comorbidity, alcoholism and states of immunosuppression) but prior antibiotic use is the dominant risk factor associated with drug-resistant S. pneumoniae.14
Epidemiologic studies have repeatedly identified recent antibiotic use as the strongest risk factor for the carriage and spread of resistant pneumococci, at both the community and individual levels. Moreover, among patients with invasive disease, recent antibiotic use correlates with an increased risk of infection with nonsusceptible (intermediate and resistant) pneumococci. This suggests not only that recent antimicrobials use increases the risk that an individual will carry, and therefore potentially transmit, resistant pneumococci, but that among infected individuals, it also increases the risk that individuals will develop invasive pneumococcal illness caused by resistant strains.16
In several countries or regions was also demonstrated that the use of specific antibiotic classes not only predisposes to resistance to that class but may facilitate resistance to unrelated antibiotic classes.
17
Those observations have led to the development of control and prevention programs that promote more judicious antibiotic use which results in reduced antibiotics prescriptions, avoiding unnecessary antibiotics (e.g., for viral infections), and in proposed few treatment modifications which include: modifying the duration of therapy, increasing treatment dosage, choosing drugs which are less effective at selecting for resistance, treating with multiple drugs and cycling of drugs at the hospital or community level.
Nevertheless despite the dramatic escalation in the rate of antimicrobial resistance among pneumococci worldwide, the clinical impact of antimicrobial resistance is difficult to define. Treatment failures due to antibiotic-resistant pneumococci have been reported with meningitis, otitis media, and lower respiratory tract infections, but the relation between drug resistance and treatment failures has not been convincingly established.
Clinical failures often reflect factors independent of antimicrobial susceptibility of the infecting organisms (host factors such as age, underlying immunosuppressive or debilitating disease and comorbidities, or intrinsic virulence of the bacteria, etc.). For this reason dissecting out the impact of antimicrobial resistance on clinical outcomes is difficult, if not impossible.
For these reasons vaccines represent the best promise to prevent both pneumococcal disease and resistance, and clinical trials have demonstrated that vaccines confer protection against invasive and some non invasive pneumococcal disease and may also protect against carriage of vaccine-included serotypes.
1.2.3
Vaccines against S. Pneumoniae
As widely described, the capsular polysaccharides are an essential virulence factor for invasive pneumococcal disease. Approximately 90% of the most frequent invasive pneumococcal diseases
belong to 23 serogroups and the 13 most common serotypes cause at least 70-75% of invasive disease in children.4
Current S. Pneumoniae vaccines are based on the use of the bacterial capsular polysaccharides (PS), which induce type specific antibodies that activate and fix complement and promote bacterial opsonization and phagocytosis. The two types of licensed vaccines are: pneumococcal polysaccharide vaccines (PPV) based on purified capsular polysaccharides, and pneumococcal conjugate vaccines (PCV) obtained by chemical conjugation of the capsular PS to a carrier protein.
The first approved Pneumococcal Polyvalent vaccine is Pneuvax 23® (1983, PPV23), obtained
through capsule bacterial lysis followed by purification, which is composed by 23 chemically and serologically distinct capsular polysaccharides of Streptococcus pneumoniae (1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F) responsible for about 90% of more dangerous illness in developed country.
Unfortunately bacterial polysaccharides have been considered classic Thymus- independent (TI) antigens, because they are able to activate an immune response eliciting B cells but not T cell (T cell independent) and in particular they have been defined TI type 2. It was because they can activate the more multireactive Splenic Marginal Zone (S-MZ) naïve B cells than the repertoire expressed among follicular B cells18 causing depletion in the peripheral memory B-cells. Indeed active S-MZ B cells
migrate to the germinal centre, where the isotype switching from IgM to IgG and a likely first step to affinity maturation occur but without formation of memory B- cells. This explains why Pneumovax® shows a rapid waning of antibody concentrations from the peak concentration measured 1 month after vaccination, and why the polysaccharide vaccines are not able to induce T- cell activation and depletion of memory B-cell pool (Fig. 1.8). 19
Furthermore studies on polysaccharide vaccine and repeated doses of PPV23 in adults and children have shown that a state of immune tolerance, or hyporesponsiveness, can develop to repeated polysaccharide vaccine antigen exposures. In fact have been reported that antibody concentrations after subsequent doses of PPV23 are similar or lower than after primary vaccination, maybe because of a depletion of memory B- cells. (Fig. 1.8)
Thus the utility of additional doses of PPV23 to sustain immunity in the elderly is limited.
Moreover since splenic marginal zone (S-MZ) B cells do not mature until the second year of life, it clarifies why purified polysaccharides contained in PPV23 are poorly immunogenic in young children.
Although PPV23 vaccine is also ineffective in immunodeficient people, and is not yet clear the effectiveness against adult IPDs. Nevertheless the administration of the 23-valent pneumococcal polysaccharide vaccine is still indicated to all those aged over 64 and people aged between 2 and 64 years with chronic diseases at high risk of invasive pneumococcal disease. 20
Figure 1.8
The polysaccharides in 23vP consist of multivalent (repeating) epitopes that are able to cross-link many B-cell receptors (BCRs) on a single cell providing a strong activation signal. Thus, the high dose of multivalent polysaccharides from 23 serotypes in 23vP may induce exhaustion of the peripheral B-cell pool by preferentially driving extra follicular proliferation of B1b, memory B, Splenic Marginal Zone B cells, and naive B cells via cross-linking BCRs, inducing a large plasma cell response. The lack of T-cell recruitment and germinal centre formation prevents replenishment of the memory B-cell pool. This is demonstrated by the reduced frequency of ASCs detected in the ELI Spot 1 month post immunization.
In 2000 a new class of anti-pneumococcal vaccine was recommended: Glycoconjugate vaccine. This new group of vaccines has been prepared by chemically controlled coupling of capsular polysaccharides (CPSs) of bacterial targets or of the oligosaccharides derived from CPSs through their reducing ends via linker molecules to carrier proteins possessing T cell peptide epitopes.
Immunization with glycoconjugate vaccines relies on the elicitation of T cell help for polysaccharide antigens, with promotion of higher affinity antibodies, immunological memory, and induces responsiveness to booster doses of vaccine, resulting in a vaccine that is both immunogenic and highly effective from early infancy.
To date belong to this group:
• Prevenar® (PCV7): the first one, contains poly- or oligosaccharides from 7 different serotypes of
S. pneumoniae (4, 6B, 9V, 14, 18C, 19F, 23F), each conjugated with genetically detoxified diphtheria toxin CRM197. The conjugation of polysaccharide antigens to a carrier protein allows obtaining an
immune response T -dependent required to induce an immunogenic reaction in children. This vaccine was effective in preventing both invasive forms of infection (meningitis, bacteremic pneumonia,
sepsis), and those not invasive, including acute otitis media and the decrease was significant even among children of superior age, adults and unvaccinated older people which are not the target of the vaccine.21 The administration of the vaccine in fact reduces the number of children carriers, which
represent the tank of pneumococci, reducing therefore also the transmission to other subjects and determining the phenomenon of “herd immunity”. It should be emphasized particularly among non-invasive infections the reduction in the incidence of cases of pneumonia and acute otitis media caused by pneumococcal vaccine serotypes.22 The use of the vaccine has finally determined also a reduction in
infections due to antibiotic-resistant pneumococcal strains.23
The introduction of vaccination with PCV7, however, has caused the greater dissemination of non-vaccine serotypes 19A, 3 and 7F, which is why recently have been produced non-vaccines directed against other 3 and 6 serotypes, respectively: the PCV10 and PCV13.
• Synflorix™ (PCV 10): has been commercialized from 2009 by GlaxoSmithKline. It uses as carrier the protein D of derivation Haemophilus influenzae for 8 of the 10 serotypes (1, 4, 5, 6B, 7F, 9V, 14 and 23F), while 18c and 19F serotypes are conjugated with tetanus and diphtheria toxoid.24
• Prevenar 13® (PCV13): from 2010 has been recommended for children (<5 years) and in
September 2011 was authorized for the IPDs prevention among adults older than 50 years. PCV13 offers more protection against rising pneumococcal serotypes as 1, 3, 5, 6A, 7F and 19A in addition to those contained in eptavalent vaccine (4, 6B, 9V, 14, 18C, 19F, 23 F) each conjugated at CRM197
detoxified diphtheria protein.
The induction of polysaccharide-specific antibodies by glycoconjugate vaccines is generated through several steps. Polysaccharide protein conjugates bind the B cell receptor (BCR) of polysaccharide-specific pre-B cells and are taken into the endosome. Once inside the cell, the protein portion is digested by proteases to release peptide epitopes, which bind to MHCII by replacing the self-peptide. The peptide from the vaccine carrier protein is presented to the αβ receptor of CD4+ T cells in the context of the MHCII molecule. Peptide/MHCII-activated T cells release cytokines to stimulate B cell maturation and induce immunoglobulin class switching from IgM to polysaccharide-specific IgG (Fig. 1.9).
Although efforts have been made to test this hypothesis at the cellular level, the precise molecular mechanisms of glycoconjugate processing and presentation in the MHCII pathway have not yet been fully dissected.25
A better understanding of these interactions is likely to facilitate the development of drugs and vaccines for the treatment and prevention of infectious and autoimmune diseases.
For this reason, synthetic vaccines could provide a useful means for the study of recognition mechanisms that are the basis of the development of a mature and well-balanced body's defence system against particular pathogens as S. pneumoniae.
In addition, alternative to glycoconjugate vaccines could reduce or even eliminate all the problems given by their heterogeneity. For this reason new efforts have been conducing to produce much more reproducible and structurally known systems on which could be possible apply modifications for obtaining vaccines much more effective and selective, minimizing side effects and resistance phenomena.
References
1 Jedrzejas, M.J. Microbil. Mol. Biol. Rev.2001, 65, 187-207.
2 Shetty, A. K.; Maldonado, Y.A. Curr. Pediatr. Rep. 2013, 1, 158-169.
3 WHO annual subscription “Weekly epidemiologica lrecord” 2007, 12, 82, 93-104. 4 WHO report “Acute Respiratory infections” 2009.
5 Muhammad, R.D.; Oza-Frank, R.; Zell, E.; Link-Gelles, R.; Narayan, V. K. M.; Schaffner, W.; Thomas, A.; Lexau, C.;
Bennett, N. M.; Farley, M. M.; Harison, L. H.; Reingold, A.; Hadler, J.; Beall, B.; Klugman, K.P.; Moore, M. R. Clin.
Infec. Dis. 2013, 56, 59-67.
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Nicolotti, N.; Ferriero, A.M.; Veneziano, M.A.; di Nardo, F.; Gliubizzi, M.D.; La Torre, G.; Saulle, R.; Zollo, A.; Reggio, P.; Mantovani, L.; Furneri, G.;Cortesi, P. “Quaderni dell’Italian Journal of pubblic Health” 2013, vol. 2, N° 4.
7 Weinberger, D. M.; Harboe, Z. B.; Sanders, E. A. M.; Ndiritu, M., Klugman, K.P.; Ru¨ckinger, S.; Dagan, R.; Adegbola,
R.; Cutts, F.; Johnson, H. L.; O’Brien, K. L.; Scott, J. A.;Lipsitch, M. Clin. Infect. Dis. 2010; 51(6):692–699.
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Zwitterionic polysaccharides
1.3.1
Zwitterionic
polysaccharides:
uncommon
capsular
polysaccharides
In 1982 Shaphiro et al.1 showed that was possible to obtain mice immunized against Bacteroides
fragilis when the animals were injected with its capsular polysaccharides, which represent the most important virulence factor, and that this immunity was conferred to T-cells.
This effect might contradict the T-independent nature of capsular polysaccharides (1.2.3 paragraph, Chapter 1.2), but exact definition of the B. fragilis polysaccharides was been essential to understand the structural basis of these important biological properties.
Chemical analysis of the B. fragilis capsule indicated its complexity, in fact upon bacterial surface were identified at least eight distinct capsular polysaccharides, two of which were particularly studied: PS A (A1 and A2) and PS B. The individuality of each polysaccharide was demonstrated by the fact that they both have different sugar analyses and immunoelectrophoretic and antigenic properties.
Each polysaccharide is composed of repeating oligosaccharide subunits and their High-Resolution NMR Spectroscopy2 revealed different sugar sequences. These oligosaccharides possess uncommon
constituent sugars with free amino, carboxyl and /or phosphate groups (Fig. 10).
In particular PS A1 is a tetrasaccharide repeating unit that has a balanced positively charged amino group and a negatively charged carboxyl group. PS B has a hexasaccharide repeating unit, including an unusual 2-aminoethylphosphonate substituent containing a free amino group and a negatively charged phosphonate group, and a galacturonic acid residue which contains an additional negatively charged carboxyl group (Fig. 1.10). The presence of opposite charged groups on the same bacterial polysaccharide is distinctly uncommon; most polysaccharides contain either neutral or negatively charged groups.
These two polysaccharides upon the capsule are able to induce the formation of abscesses activating nonimmune CD4+ and CD8+ T cell in the host,3 while in purified form are responsible for
Figure 1.10
It was demonstrated that PS A1 has more stimulatory effects in comparison to PS B,4 but for each
the presence of both opposite charged groups and the density of these for repeating unit are fundamental to provide both abscess induction and T cell activation against Intra-Abdominal abscess formation.5,6,7
In fact, N-acetylation or transformation of the amino group to a tertiary amine respectively abrogate or reduce the effects thus pinpointing the importance of the free amino group which could be able to form reversible bonds (known as Schiff bases) that are necessary for antigen-specific T-cell activation.8 Furthermore introduction of other free amino groups inside the structure creating a net
positive charge reduces activity of these saccharides. Reduction of the carboxyl group on the galacturonic acid of PSB creates a polysaccharide with a free amino group and one phosphonate group per repeating unit and this modification does not alter the ability of PS B to induce abscess. Indeed the oxidation of PS A1 with NaIO4 of the side chain sugar of the arabinofuranose residue creating an
aldehyde group at C-5 eliminates the capability to elicit a response, which after regeneration by NaBH4
restores the proliferative activity. (Fig. 1.11) Figure 1.11
Similar studies were also conducted on Morganella morganii9 (Fig. 1.12), another commensal gram
positive bacterium producing an antigen which repeating unit contains free amino group, second phosphate and a phosphocholine group. Tests conducted on different selectively protected derivatives (for example after acetylation of the free amino group) suggest that the zwitterionic phosphocoline
group might be sufficient for reactivity with MHCII molecules and the downstream T-cell activation such as was demonstrated for PS B. Further studies with carbohydrates are needed to explore this possibility.
Figure 1.12
To date, many other similar Zwitterionic microbial polysaccharides such as PS A2 of Bacteroides fragilis, Streptococcus pneumoniae type 1CP, Staphilococcus aureus type 5CP and 8CP10 (Fig. 1.12) and
synthesized peptides to mimic the charged motif of Zwitterion polysaccharides (Lysine-aspartic acid peptides with more than 15 repeating units) have been tested to evaluate their ability to induce abscess or to activate T-cells. In vitro experiments have showed that these compounds form protective agents against abscess formation.
These findings strongly suggest that ZPSs are a novel class of T-cell antigens and possess exceptional immunological activities. They are recognizable by both B and T lymphocytes and thus have the ability to modulate the dual arms of the host immune system, revising the conventional dogma that carbohydrates are T-cell independent antigens.
1.3.1.1
Structural analysis
An understanding of how ZPSs differ from other carbohydrates and exert their peculiar effects on T cells will provide important new insights into the structure-function relationship of carbohydrates and the immune system.
T-cell-activating ZPSs vary significantly in their monosaccharide compositions, linkages, and sequences. The only obvious common feature is their zwitterionic charge motif which is generally rare among naturally occurring polysaccharides but functionally critical for ZPSs. Several studies for explaining why ZPSs, comprising different carbohydrate sequences, elicited similar T-cell responses
