ARI-WHO

Acute Respiratory Infections (Update September 2009)

Introduction

Respiratory disease burden

Acute respiratory infections (ARIs) continue to be the leading cause of acute illnesses worldwide and remain the most importantcause of infant and young children mortality, accounting for about two million deaths each year [1] [2] [3] and ranking firstamong causes of disability-adjusted life-years (DALYs) lost in developing countries (94.6 millions, 6.3% of total [4]. Thepopulations most at risk for developing a fatal respiratory disease are the very young, the elderly, and the immunocompromised.While upper respiratory infections (URIs) are very frequent but seldom life-threatening, lower respiratory infections (LRIs) areresponsible for more severe illnesses such as influenza, pneumonia, tuberculosis, and bronchiolitis that are the leadingcontributors to ARIs' mortality [5]. Pneumonia, with a global burden of 5 000 childhood deaths every day, is a tangible threat thatneeds to be dealt with accordingly.

The incidence of ARIs in children aged less than 5 years is estimated to be 0.29 and 0.05 episodes per child-year in developingand industrialized countries, respectively, which translates into 151 million and 5 million new episodes each year, respectively[6]. Most cases occur in India (43 million), China (21 million), Pakistan (10 million), Bangladesh, Indonesia and Nigeria (56million each). Pneumonia is responsible for about 21% of all deaths in children aged less than 5 years, leading to estimate that ofevery 1000 children born alive, 12-20 die from pneumonia before their fifth birthday [4]. .

The main etiological agents responsible for ARIs in children include Streptococcus pneumoniae, Haemophilus influenzae type b(Hib), Staphylococcus aureus and other bacterial species, respiratory syncytial virus (RSV), measles virus, human parainfluenzaviruses type 1, 2, and 3 (PIV-1, PIV-2 and PIV-3), influenza virus and varicella virus.

Bacterial diseases

S pneumoniae (pneumococcus) was identified in 30%-50% of bacterial pneumonia cases in developing countries in the 1990s,followed by Hib (10%-30% of cases), then Staphylococcus aureus and Klebsiella pneumoniae [6] . Non-typable H influenzae(NTHI), and non-typhoid Salmonella spp have also been implicated in some but not all studies. Other organisms, such asMycoplasma pneumoniae, Chlamydia spp, Pseudomonas spp and Escherichia coli also can cause pneumonia. The most commonsyndromes associated with M pneumoniae infections are acute bronchitis, pharyngitis and otitis, but 10% of infected childrendevelop pneumonia [7].

The introduction of Hib conjugate vaccines has resulted in a truly remarkable decline in Hib disease where the vaccine has beenintroduced. However, the vaccine is not yet routinely made available to a majority of children worldwide. As a result, 400 000deaths are still estimated to occur from Hib disease each year [8]. In view of their safety and remarkable efficacy, the WHOhas recommended the global implementation of the Hib conjugate vaccines [9].

S pneumoniae is estimated to cause more than one-third of the 2 million deaths due to ARIs, especially in developing countrieswhere the bacterium is one of the most important bacterial pathogens of infancy and early childhood [10] . Virtually every childin the world is colonized with one or more strains of pneumococcus and becomes a nasopharyngeal carrier during his first fewyears of its life. Many children will go on to develop otitis media, and a few will eventually develop invasive pneumococcaldisease including bacteremic pneumonia and/or meningitis [11] . The introduction of the conjugate pneumococcal vaccine inroutine infant immunization should have a major impact on pneumonia in children less than five years of age worldwide [12] , asalready documented in the USA [13] .

Last but not least, tuberculosis (TB) continues to be a leading cause of deaths worldwide, with an estimated one third of humanityinfected and about 1.7 million deaths each year, a global toll of 4650 lives daily. The emergence of Mycobacterium tuberculosis

WHO | Acute Respiratory Infections (Update Sep... http://www.who.int/vaccine_research/diseases/ari/...

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(Mtb) strains carrying drug-resistance mutations against first-line dugs (MDR-TB) and, more recently, against both first- andsecond-line drugs (XDR-TB), shows that it will most probably be impossible to contain the TB pandemic with drugs alone. Morethan one hundred new TB vaccine candidates have been tested in animal models and some have moved into clinical trials. Testingsuch a wide variety of vaccine types using different strategies will obviously require time and a lot of coordination, especially assurrogate markers of protection still remain mostly unknown at this time.

Viral diseases

Among the viral agents of ARIs, measles virus was still responsible, in 2002, and in spite of the inclusion of the live attenuatedmeasles vaccine in the Expanded Program of Immunization (EPI), of some 213 000 deaths worldwide, essentially due toinsufficient vaccine coverage [14] . The situation has fortunately been substantially improved lately [15] .

But the leading cause of serious respiratory illness in young children is respiratory syncytial virus (RSV), the agent of infantilebronchiolitis, which is associated with substantial morbidity and mortality [16] . Parainfluenza viruses (PIV-1, PIV-2 andPIV-3), especially PIV-3, are second in incidence immediately after RSV. All children by the age of 2 years have had at leastone episode of PIV and/or RSV illness. In addition, both viruses can cause severe disease in the elderly, especially in patientswith a chronic respiratory or cardiac condition. Although the disease burden due to these pathogens has not been accuratelyquantified in developing countries, extrapolation from known figures in industrialized countries, such as 125,000 reported casesof RSV per year in the USA [17] , leads to the impressive global estimates of 64 million cases and 160,000 deaths per year fromRSV infection worldwide. RSV was identified in 15-40% of pneumonia or bronchiolitis cases admitted to hospital in developingcountries, followed by influenza viruses, parainfluenza viruses, human metapneumovirus and adenovirus [18] . The elderly alsoare at risk for severe RSV disease, and 14 000 to 60 000 RSV-related hospitalizations of the elderly are reported to occurannually in the USA [19] .

Human metapneumovirus, a member of the Paramyxoviridae family, is a recognized cause of a large fraction of severe ARIs ininfant, elderly and immunocompromised population [20] [21] [22] [23] Other viruses that cause respiratory infections arecoronaviruses, adenoviruses and rhinoviruses. Recently discovered coronaviruses HCoV-HKU1 and HCoV-NL63 are significantpathogens that contribute to the hospitalization of children for ARI [24] [25] [26] . Among other members of the Coronaviridaefamily are human coronaviruses HCoV-229E and HCoV-OC43, agents of the common cold, the feline infectious peritonitis virus(FIPV), the avian infectious bronchitis virus (IBV) and the pig transmissible gastroenteritis virus (TGEV).

Another recently identified coronavirus is that of the severe acute respiratory syndrome (SARS), SARS-CoV, which emerged insouthern China in late 2002 and spread in the spring of 2003 to some 30 countries within Asia, Europe and North America [27][28] . The epidemic finally came to a stop in July 2003 through strict implementation of quarantine and isolation procedures andthanks to international collaboration under the coordination of WHO [29] . At that date, 8,096 cases had been identifiedworldwide and 774 patients had died, a 9.6% mortality rate. SARS-CoV belongs to a newly identified group in the familyCoronaviridae, which are enveloped RNA viruses whose envelope is characterized by crown-like proteinic spikes, and whosegenome is an exceptionally long 29 727 nucleotides single-stranded positive RNA molecule that encodes 23 different proteins.The viral spike (S) protein is responsible for the induction of virus neutralizing antibodies [30] [31] [32] .

Although there is evidence that SARS-CoV emerged from a nonhuman source, no animal reservoir has yet been identified withcertainty. Masked palm civet cats and raccoon dogs have been found to be carriers of the virus [33] [34] , and Chinesewild-animal traders, especially civet cats traders, showed high seroprevalence figures. Horseshoe bats carry a SARS-CoV-likevirus and might be the virus reservoir [35] [36] [37] . Less than one year after SARS first appeared, half a dozen candidatevaccines already were in development [38] , including whole inactivated virus vaccines, live recombinant vector-based vaccines,VLPs, and subunit vaccines. More recently, a live attenuated virus has been developed [39] . Most of these efforts have howeverbeen put on hold, in view of the current elimination of the disease.

[40] [41] In the elderly, influenza-related pneumonia remains a leading cause of infectious disease-related deaths. The threat ofan avian influenza pandemic has been looming ever since the emergence in 1997 in Hong Kong of the H5N1 avian influenzavirus, especially since the reappearance of human cases in 2003-2004. The new H5N1 variant is highly pathogenic for poultryand wild birds and can lethally infect cats and humans. At this time, however, it still is not possible to predict which virus isgoing to eventually cause a pandemic and when it is going to happen [42] [43] , but the preparation of pandemic influenzavaccines is being actively pursued, generating broad new knowledge on how to possibly improve seasonal influenza vaccineimmunogenicity [44] [45] .

Regarding influenza virus, the average global burden of inter-pandemic influenza may be on the order of 1 billion cases per year,leading to 300,000-500,000 deaths worldwide Viruses are a common cause of ARIs in children worldwide. However, the


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substantial reduction in ARI mortality observed in developing countries that have implemented simple case management includingprovision of antibiotics to children with ARI suggests that bacterial pneumonia contributes to a large proportion of deaths in thesepopulations [46] . Available data suggest that dual infections with viral and bacterial pathogens may be quite common, as seen bythe fact that, in the industrialized world, epidemics of RSV and/or influenza coincide with epidemics of S pneumoniae year afteryear [47] . While influenza virus is the most commonly met pathogen in this context, other respiratory viruses, including RSV,measles virus, parainfluenza viruses, or adenoviruses may also predispose to secondary bacterial infections. Several differentbacterial species may be implicated, including H influenzae, Staphylococcus aureus, Streptococcus pyogenes, Mycoplasmapneumoniae, and, above all, S pneumoniae [48] . Half or more of the flu-associated mortality in the 1918-1919 Spanish Fluepidemic is believed to have resulted from pneumococcal superinfections [49] [50] .

The same is true for developing countries. As an example, the observation was made in South Africa that children vaccinatedwith the 7-valent conjugate pneumococcal vaccine showed 31% reduction in virus-associated pneumonias requiringhospitalization, strongly emphasizing the presumed importance of dual infections involving S pneumoniae [51] . Dual infectionseems to increase the severity of the disease and to result in higher mortality. This might be due to inhibition of pulmonaryantibacterial defenses during recovery from viral infection [52] .

Finally, it should be emphasized that nosocomial or hospital-acquired pneumonia is a major public health problem: pneumonia isthe second most common type of all nosocomial infections, with an associated case fatality rate of 20%-50%.

Influenza

- Introduction- Disease Burden- Virology- Vaccines

- Meetings reports- Other WHO Links- H1N1

Introduction

The repetitive occurrence of yearly, seasonal influenza epidemics is due to the fact that influenza viruses are continuouslychanging antigenically. This was well illustrated by the emergence of the influenza A H3N2 'Fujian' strain, which appeared inJuly 2003 in the southern hemisphere then spread to the northern hemisphere a few months later to become a dominant strain [53]. To face this continuous change, virus strains to be included in the vaccine are updated annually so as to match the circulatingvirus strains. An alternative would be to develop "universal" influenza vaccines which would cover all possible influenzavirusstrains (see below). Meanwhile, the recent emergence of highly pathogenic avian influenza H5N1 in poultry farms and marketsin several countries in Asia, from where it spread to Africa and Europe, and its transmission to humans with a 60% case fatalityrate, has revived the fear of a possible new pandemic of influenza with high mortality and planetary consequences [33] [54] [55].

Disease Burden

Seasonal influenza

The burden of influenza is currently estimated to be 25-50 million cases per year (~ 20% of the population) in the USA alone,leading to 150 000-200 000 hospitalizations and 30,000-40,000 deaths [56] . If one extrapolates these figures to the rest of theworld, the average global burden of seasonal influenza comes to be on the order of 600 million cases, 3 million cases of severeillness and 250 000-500 000 deaths per year. Hospitalization rates from severe illness can be as high as 3 per 1000 for 6 to 23months old children and as high as 9 per 1000 for children younger than 6 months [57].

Influenza is highly contagious [58] and is readily transmitted via aerosols and droplets from the respiratory tract of infectedpersons by direct contact, through coughing or sneezing, or by hands contaminated with respiratory secretions. Adults are mostinfectious from 1 day before symptom onset up to 7 days afterwards. When influenza is introduced into a household, 20-60% ofexposed persons will eventually show virologic or serologic evidence of infection. The disease can affect all age groups, butrates of infections are highest among young children who shed virus and are a potential source of infection in older age cohorts


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[59] , whereas rates of serious illness, complications and death are highest in persons aged 65 years and older, as well as inpersons with chronic cardiac or respiratory conditions. Data collected in Michigan (USA) in Japan and in Russia indicate thatmass vaccination of school-aged children correlates with a reduced rate of respiratory illness in all age groups, suggesting thatlarger-scale immunization in childhood could favourably affect influenza epidemics [60].

Epidemics and outbreaks of influenza occur in different seasonal patterns depending on the region in the world: in temperateclimate zones, seasonal epidemics typically begin in the late Fall and peak in mid-winter, infecting about 5-15% of the populationeach season. In tropical zones, seasonal patterns are less pronounced and the virus can be isolated year-round [61] . The disease,characteristically a febrile illness with respiratory symptoms, ranges in severity from mild to debilitating and can lead to lethalprimary fulminant pneumonia, particularly in persons with underlying pulmonary or cardiopulmonary pathologies.

The repetitive occurrence of yearly influenza epidemics is maintained through the ongoing process of "antigenic drift", whichresults from the accumulation of point mutations in the genes that encode the two viral surface proteins haemagglutinin (HA) andneuraminidase (NA), and leads to the constant emergence of new virus variants against which there is little or no pre-existingimmunity in the population. For this reason, major seasonal epidemics of influenza continue to occur each year and the virusstrains to be included in the vaccine of the year must be chosen to match the emerging new variants [40] [62] Recent molecularstudies suggest that new virus strains emerge in East and South-East Asian countries, from which they spread around the world,first to Europe and North America which are reached within 6-9 months then to South America [63].

Pandemic influenza

At unpredictable intervals, due to the segmented nature of the influenza virus genome, circulating human influenzavirus A strainsalso can acquire new genes from an avian or other animal influenza virus. This process is believed to occur most readily in pigs,as pigs have the complete set of syalilated receptors for avian, swine and human influenza virus strains. Co-infection in pigs canthus result in the emergence of a virus with a completely new glycoprotein subtype, which is referred to as an "antigenic shift".If the reassortant virus can efficiently spread into the human population, a worldwide pandemic can occur, as was the case in1918, 1957, and 1968 [43] [64].

The impact of a new influenza pandemic has grossly been estimated at 1-2 billion cases of flu, 5-12 million cases of severeillness, and 1.5-3.5 million deaths worldwide [65] . It could result in 1 million to 2.3 million hospitalizations and 250 000 to 650000 deaths in industrialized nations alone. In the USA, the impact of a new pandemic, assuming it would be of a similarmagnitude as the 1957 or the 1968 pandemics, and not like the 1918 pandemic, is projected to be 18-42 million outpatient visits,314 000-734 000 hospitalizations and 89 000-207 000 deaths. Its impact would be even more devastating in developing countries[66] . Historians estimate that more than 50 million people died in the 1918-1920 influenza pandemic [67] [68].

Of note is the observation that in three studies of blood cultures taken from living soldiers with pandemic influenza-associatedpneumonia in 1918, pneumococci were isolated from 46% of patients. The data from influenza hospitalization in children andfrom pandemic influenza mortality in 1918 suggest a significant role for pneumococci in influenza-associated pneumonia [69] .This is most likely due to inhibition of macrophage-mediated antibacterial defense during the recovery stage from influenzainfection, which is characterized by pulmonary infiltration of T cells that secrete high amounts of IFN-? [52] . This suggests thatthe prevention of pneumococcal super-infection should be an essential part of pandemic influenza preparedness [70] .

The emergence of the avian H5N1 influenzavirus A strain with pandemic potential occurred in 1997 in Hong Kong, SAR,resulting in the death of 6 of the 18 affected patients, mainly young adults [71] . The virus was fortunately not able to spreadfrom person-to-person and the outbreak was controlled through massive culling of poultry. The H5N1 strain however reemergedin 2003-2004 in China, Japan, South Korea then Thailand, Vietnam, Indonesia, Cambodia and Malaysia, leading to massiveculling and attempts at vaccination of poultry. More than 60% of human patients diagnosed with the virus died. In May, 2005, ahighly pathogenic (HP) H5N1 variant emerged in wild birds in Quinghai Lake, China [72] [73] [74] , that not only killeddomestic poultry but also wild aquatic birds. The HP strain also is pathogenic for ferrets, cats and tigers. Cats can be infectedboth by the respiratory route and by feeding on virus-infected birds. The HP H5N1 strain spread to a great many countries inAsia, Africa and Europe where it was repeatedly recovered from migratory birds and was the cause of multiple outbreaks inpoultry [75] . A significant part of transmission has been linked to commercial trade of poultry and derived products [76] .

Cases of human H5N1 infections have been reported in many countries but no human-to-human transmission has been evidencedso far, except in a couple of instances among close contacts. As of October, 2008, a total of 387 confirmed human cases from 15countries resulting in 245 deaths had been reported to WHO, of which more than 100 cases and 50 deaths had occurred inVietnam. The virus seems to now have become endemic in several countries, including Nigeria, Egypt, Bangladesh, Vietnam andIndonesia, while showing continuous sequence evolution leading to the emergence of different molecular clades and sub-clades


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[77] . Significant concerns therefore remain that the virus could adapt to infect humans or exchange genetic material withco-circulating human influenza viruses, resulting in the emergence of a highly pathogenic pandemic strain [78] .

Other avian influenza viruses have occasionally caused a human outbreak, such as a H9N2 strain in 1999 in Hong Kong, a H7N7strain in 2003 in the Netherlands, which caused 89 confirmed human cases with conjunctivitis and one death, and H7N2 andH7N3 in 2003-2004 in North America. No one can know how severe the next pandemic will be, nor which influenza virus willcause it -it could be an H2, H5, H7, H9 or another subtype. A report from the Centers for Disease Control and Prevention(CDC) indicates that North American H7N2 and H7N3 strains have now partially adapted to the human sialic acid receptor (seebelow), a characteristics that would enhance the virus potential to infect and spread among humans [79] . At the same time, theH5N1 virus continues to be a zoonotic virus, not a human-adapted one, and human infections remain rare. Still, given thealarming 60% average case fatality rate of H5N1 influenza cases in humans so far, it probably is prudent to prepare for the'worst case' scenario [78] .

Virology

Influenza viruses are enveloped viruses with a segmented genome made of eight single-stranded negative RNA segments, mostof which encode only one viral protein (for a review, see [80] ). They form the family Orthomyxoviridae, which includes threegenera, Influenzavirus A, Influenzavirus B, and Influenzavirus C, that differ antigenically by two of the structural proteins, thematrix (M) protein and the nucleoprotein (NP). Influenza A viruses are further divided into subtypes according to theantigenicity of their major envelope glycoproteins, the haemagglutinin (HA) and neuraminidase (NA). Sixteen HA subtypes (H1to H16) and nine NA subtypes (N1 to N9) have been identified. The nomenclature of human influenza virus strains includes thetype of the isolate, the place where it was isolated, the year of isolation, an identification number and for influenza A viruses,the subtype of both HA and NA, e.g. "A/Panama/2007/99 (H3N2)" [40] .

Influenza A viruses of the H1N1, H1N2, H3N2 and H2N2 subtypes naturally infect humans, but only the first three types arecurrently circulating in the human population. In contrast, influenza A viruses of all 16 HA subtypes and all 9 NA subtypes havebeen recovered from aquatic birds, which serve as a natural virus reservoir and a potential source of new genes for pandemicinfluenza viruses. Study of the epidemiological dynamics of human influenza A virus shows that genomic segments encoding thenon-structural proteins (NS1 and NS2), the matrix proteins (M1 and M2), and two of the polymerase proteins, PB2 and PA, haveremained unchanged since the global pandemic of 1918 [81] [82] . In contrast, new RNA segments encoding the haemagglutinin(HA) and neuraminidase (NA) glycoproteins, as well as the PB1 polymerase, have been acquired through reassortment withavian influenza virus, coinciding with the global pandemics in 1957 (H2N2) and 1968 (H3). Seasonal epidemics of influenza Avirus since 1968 have been dominated by H3N2 viruses and characterized by punctual mutations, natural selection and frequentreassortment that underlie antigenic drift [83] [84] [85] .

Influenza A viruses also can infect poultry, pigs, horses, dogs, and sea mammals. Interspecies transmission has been welldocumented [86] . Aquatic birds, in which the virus multiplies in the gut, usually have an asymptomatic infection and excrete thevirus in their faeces. They can transport the virus over large geographical distances. Influenza B viruses appear to naturallyinfect only humans, although infection of seals was documented in the Netherlands [87] . Influenza C viruses only appear toinfect humans and pigs and usually cause sporadic cases of upper respiratory disease.

The HA glycoprotein molecules are present at the surface of the influenza virion in the form of a trimeric HA0 precursor whichmust undergo proteolytic cleavage to generate functional subunits HA1 and HA2. HA1 bears the receptor-binding site andneutralization epitopes, whereas HA2 is responsible for the fusion of the viral envelope with the host-cell membrane at acidicpH [88] [89] [90] [91] . Classical avian virus strains have a HA0 cleavage site which is trypsin-like, hence their tropism for thegastrointestinal tract. In contrast, highly virulent avian strains such as the 2004 H5N1 strains from Thailand and Viet Nam or theHP H5N1 isolates have acquired through spontaneous mutations an ubiquitous furin-like cleavage site, which allows them tomultiply in many tissues including the respiratory tract [92] . A recent post-mortem study of two fatal H5N1 human casesshowed that in addition to the lungs, the virus had disseminated to other organs including the intestine, T lymphocytes in lymphnodes and circulating monocytes and macrophages, as well as cerebral neurons [93] . The virus could also be transmitted frommother to foetus across the placenta. Viremia and extra-respiratory complications appear to be more common with the avianH5N1 virus than with usual human influenzaviruses [94] .

Influenza virus entry into cells of the respiratory tract is mediated by binding of the trimeric haemagglutinin spikes on the virionto specific sialic acid receptors at the cell surface. The difference between the avian (H5) and human (H1, H3) hemagglutininslies in their specificity for ?-2,3-Gal or ?-2,6-Gal sialic acid linkages, respectively [95] . In mammals, cells with ?-2,3 receptorsonly occur deep in the lungs, whereas ?-2,6 receptors are found in the upper respiratory tract, explaining why the H5N1influenza subtype may cause infection of the lower respiratory tract and severe pneumonia in humans but does not spread readily


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from human-to-human [96] . Two amino acid mutations that caused a switch in receptor binding preference from the human?-2,6 to the avian ?-2,3 sialic acid resulted in a 1918 H1N1 virus incapable of respiratory droplet transmission between ferrets,suggesting that a predominant ?-2,6 sialic acid-binding preference is essential for optimal transmission of influenza virusesamong mammals [97] . Three mutations in the H5 haemagglutinin were both necessary and sufficient to change its receptorbinding specificity from the avian ?-2,3 to an human ?-2,6 pattern [98] .

The pathogenicity of influenza A virus is clearly due to a polygenic effect [99] [100] . Studies of virulence of avian influenzavirus strains shows that in addition to the HA cleavage site (see above), changes in NS1 are important determinants [100] [101] ,as well as mutations in the PA and PB1 genes [102] . Similarly, the HA, NS, NP, PA, PB1 and PB2 proteins contribute to thevirulence of influenza A virus strains in mice, pigs and monkeys (reviewed in [99] ). The NS1 protein is critical for thepathogenicity of H5N1 influenza viruses in mammalian hosts as it antagonizes host cell interferon induction and the double-stranded RNA-mediated activation of the NF-kappaB and IFN pathways [103] [104] [105].

Vaccines

The currently available influenza vaccines are made from either inactivated, detergent-split or whole virion influenza virus orlive attenuated influenza virus (LAIV) propagated in the allantoic cavity of 9-12 days embryonated chicken eggs from certifiedfarms under strict veterinary control [106] . They include two currently circulating influenza A virus strains and one influenza Bvirus strain. These trivalent vaccines, which have been used for decades in industrialized countries to prevent seasonal influenzainfection, provide a high benefit/cost ratio in terms of preventing hospitalizations and deaths, as shown in numerous studies onvaccination of the elderly and of individuals at high risk for severe outcomes of influenza [40] . Recommended recipients ofinfluenza vaccines are people at high risk of influenza-related complications, namely adults and children with chronic healthconditions such as cardiac or pulmonary disorders, asthma, cancer, immunodeficiency or immunosuppression, people who areresidents of nursing homes and other chronic care facilities, people over 65 years of age, healthy children aged 6-23 months[107] [108] and pregnant women. Household contacts of individuals at high-risk, as well as providers of health care in facility orcommunity settings who are potentially capable of transmitting the virus to vulnerable populations should also be vaccinated, aswell as those providing regular child care to children under 24 months of age.

WHO estimates that there globally are about 1.2 billion people at high risk for severe influenza outcomes: 385 million elderlyover 65 years of age, 140 million infants, and 700 million children and adults with an underlying chronic health problem. Inaddition, 24 million health-care workers ought to be immunized to prevent spreading of the disease to the high-risk population.

Seasonal inactivated vaccines.

Continual antigenic drift of the virus means that a new vaccine updated to contain the most current circulating strains is neededevery year to protect against new seasonal infections. This is done by reassortment or through the use of the techniques ofreverse genetics, which allow one to transfer the HA and NA genomic segments from a circulating wild-type virus into a wellcharacterized, high-yield egg-adapted or cell-culture-adapted influenza virus strain such as the PR8 virus strain, then to selectthe reassortant virus with the desired HA and NA gene combination. The reassortant virus is usually grown in the allantoic cavityof embryonated hen's eggs, although some vaccines are now produced using mammalian cell lines such as MDCK, PERC-6 orVero cells.

Monovalent vaccine lots are inactivated using either formalin or ?-propiolactone, then purified and combined to make the finaltrivalent vaccine [109] [110] . Most seasonal vaccines produced today are 'split-vaccines' (subvirion vaccines), which result fromtreatment of the virions with a detergent or a solvent to dissolve the viral lipid envelope. Less frequently used are whole-virionvaccines made of intact inactivated virus, which may be more reactogenic but have consistantly proved to be better immunogensand confer more efficient protective responses than split vaccines [111].

Other inactivation procedures have been reported, such as treatment with hydrophobic 1,5-iodonaphtyl-azide (INA), whichselectively targets the transmembrane segments of proteins in the viral envelope while preserving the structure of the antigens onthe surface of the virus. INA is activated by UV irradiation, resulting in a non-infectious intact influenza virus particle with fullneuraminidase activity able to induce potent anti-influenza virus serum neutralizing antibody and T cell responses and to provideprotection against heterosubtypic challenge after a single Intranasal immunization, similar to live attenuated virus immunization[112].

The trivalent split inactivated influenza vaccines have a remarkable safety profile, including in 6 to 23 months old children [113], as recently confirmed in a retrospective study bearing on 45,356 vaccinations in children that age [108] . Multiple studies show


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the vaccine to be approximately 60% to 90% efficacious against influenza illness in healthy children and adults, depending on theantigenic match between the circulating and vaccine viral strains [114] [115] [116] . Vaccine effectiveness was found to actuallyvary substantially across successive seasons from a low 10% to a high 52% depending on the antigenic match between thecirculating virus strains and the strains used in the vaccine [117] . Vaccination was also found to decrease the incidence ofpneumonia, physician visits, hospitalizations and deaths in the elderly [118] [119] [120] . In vaccine-naive children less than 9years old, a two dose schedule is required [121] [122] . In contrast, a second dose of vaccine in elderly individuals does not boostimmunity [123].

The inactivated split influenza vaccine also is quite immunogenic when administered as a full dose (15µg HA) to elderlyvolunteers by the ID route using a Beckton-Dickinson microneedle delivery system [124] [125] . The use of the ID route wasfound to enhance the immunogenicity of the vaccine in the elderly population as compared to the classical IM route ofadministration, as judged by higher geometric mean antibody titers and higher percentage of vaccinees with a > 4-fold rise in HAantibody titer [126] . Similar data were generated in recipients of renal transplants. A randomized, open-label study in 112healthy children aged 3 to 18 years showed that the immunogenicity of ID vaccination at one fifth of a dose was comparable tothat of standard-dose IM vaccination in children as young as 3 years of age [127] . Attempts at vaccinating healthy adults by theintranasal route using the inactivated split vaccine in combination with detoxified E coli enterotoxin (LTK63) as an adjuvant alsoare in progress [128] [129].

At this time, the vaccination of people at high risk of complications from influenza is a key public health strategy inindustrialized countries. Almost 350 million trivalent vaccines are distributed worldwide each year. Universal influenzavaccination has been proposed as one strategy to improve vaccination coverage and disease prevention. Most economic analysesshow that universal vaccination of healthy adults and children would be cost-effective. Also, herd immunity resulting fromvaccination of school-age children would substantially reduce the incidence of disease and mortality among adults [129] [130] .The world's total vaccine production capacity at this time, however, is only about 350 million doses of trivalent vaccine, of whichclose to 50% are produced by just one manufacturer, Sanofi Pasteur.

Live attenuated influenza vaccines (LAIVs)

The second major approach to influenza vaccines has been the development of cold-adapted (ca) virus strains which grow well inprimary chicken kidney cells and embryonated eggs at 25-33°C, have a reduced replication titre at 37°C, and show attenuatedvirulence in ferrets. Cold adaptation was found to be a reliable and efficient procedure for the derivation of LAIVs, which areobtained by reassorting the HA and NA genomic segments from a circulating influenza A virus strain with the other six genomicsegments from a well characterized laboratory strain that carries the ca mutation. Thus, a bivalent ca LAIV was developed byMicrogen and used for decades in Russia to immunize more than 100 million people every year.

A trivalent ca LAIV (FlumistTM) has been developed for intra-nasal spray delivery by MedImmune and Wyeth in the USAwhere it officially is licensed for 2-59 years-old persons. The FlumistTM vaccine was proven highly efficacious in Phase IIItrials, showing a 92% overall protection rate over a 2-year study in children [131] [132] . It was well tolerated and effective inpreventing culture-confirmed influenza illness in children as young as 6 months of age who attended day care centers [133] andwas shown to provide herd immunity to adults when used in children [134] . It also demonstrated superior efficacy compared tothe trivalent inactivated vaccine in preventing influenza illness in children 12 to 59 months of age [135] , in young children withrecurrent respiratory tract infections [136] and in children with asthma [137] [138] . One dose of FlumistTM induced significantprotection against influenza illness and pneumonia in 5 to 18 years old children when administered during an influenza outbreakdue to a circulating influenza virus strain distinct from the vaccine strain, whereas the trivalent inactivated vaccine was not ableto provide protection [139] . Nasal shedding of LAIV in individuals 5-49 years of age was found to generally be of short duration(days 1-10 post immunization) and at low titers. LAIV recipients should therefore avoid contact with severely immunosuppressedpersons [140].

The FlumistTM vaccine shows remarkable genetic stability, but it has to be kept at -18°C. A new, heat-stable version of thevaccine has been developed, which showed good efficacy in clinical trials in Asia and Europe, but was associated with anincrease in asthma episodes in young children [141] . The current vaccine was also tested for safety in infants aged 6 to 24weeks, who received two doses of vaccine intranasally 1 month apart: the vaccine was generally well tolerated [142] . Inchildren, LAIV provided sustained protection against influenza illness caused by antigenically related strains for 9-12 months,and showed meaningful efficacy although at a lesser level through a second season without revaccination [143].

Another ca LAIV also is in development at Applied Microbiology in Austria by growth of the virus in Vero cells at 25 °C [144] .Still another cold-adapted LAIV grown in Madin-Darby canine kidney (MDCK) cells on microcarrier beads in serum-freemedium is at an advanced preclinical development stage at the Vector Scientific Center in the Russian Federation.


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Preliminary studies indicate that the IFN?-ELISPOT assay, a measure of cell-mediated immunity, may be a more sensitivemeasure of influenza immune memory responses to LAIVs than serum antibody. The role of cell-mediated immunity in actualprotection against culture-confirmed clinical influenza by LAIV was investigated in a large efficacy trial involving 2172 6 to 36months-old children in the Philippines and Thailand who were vaccinated with an intranasal dose of 107 FFU of LAIV: themajority of infants with >100 IFN-? spot-forming cells/106 peripheral blood mononuclear cells were protected against influenza[145].

Other types of influenza vaccines

Virosomes

Berna Biotech, now Crucell, is commercializing an influenza vaccine formulated in virosomes, with the surface spikes of thethree currently circulating influenza virus strains inserted into the vesicle membrane of liposomes. A nasal formulation of thevaccine had to be withdrawn from the market, due to undesirable neurological side effects (Bell's palsy) linked to the presenceof the E. coli labile toxin (LT) used as an adjuvant, most likely because the B subunit of LT could bind to GM1 gangliosidereceptors in neuronal tissues associated with the olfactory tract. Other formulations of inactivated influenza vaccine for mucosaldelivery are in progress including immunostimulating complexes (ISCOMs).

Synthetic vaccines

Yeda, an Israeli R&D company, is developing a synthetic peptide influenza vaccine for nasal administration. The vaccine hasshown protective efficacy in humanized mice and is planned to enter clinical trials soon.

M2e-based vaccines

A "universal" human influenza virus A vaccine was initially developed at the University of Ghent, Belgium, based ontransmembrane viral protein M2, which is scarcely present on the virion but is abundantly expressed on virus-infected cells [146]. The extracellular M2 domain, M2e, a 23 amino acid-long peptide, is remarkably conserved between H1N1, H2N2 and H3N2influenza A virus strains [147] . Passive administration of anti-M2e antibodies affords significant protection against influenza Avirus challenge in animal models. The mechanism is believed to be NK cell-mediated antibody-dependent cellular cytotoxicity(ADCC), not virus neutralization.

A recombinant particulate vaccine has been engineered by genetically fusing copies of the influenza virus M2e domain to thehepatitis B core antigen (HBc). The (M2)-HBc fusion protein was found to spontaneously assemble into highly immunogenicvirus-like particles (VLPs) that provided complete protection against a lethal influenza A virus challenge in mice and ferrets[148] . The VLP vaccine, ACAM-FLU-ATM, which is developed by Acambis, Cambridge, MA, USA, was recently tested in atwo-dose immunization schedule clinical study in 18-40 years-old volunteers.

Another formulation involving a fusion protein based on the CTA1-DD adjuvant and containing tandem repeats of the M2esequence, still is at a preclinical stage of development [149] [150] . These vaccines could theoretically serve as universalinfluenza A vaccine in view of the high degree of conservation of the M2e sequence among human influenza A viruses. Thequestion of whether they would also protect against a pandemic virus is however open, as the M2e sequence in avian virus strainsincluding H5N1 appears to be divergent from that in humans viruses.

The M2e peptide has also been conjugated to other protein carriers, such as the Neisseria meningitidis outer membrane proteincomplex (OMPC) and derivatives of the cholera toxin. Another approach has been developed at VaxInnate, USA, linking fourtandem copies of M2e to Salmonella typhimurium flagellin type 2 protein, a potent TLR5 ligand. The resulting STF2.4xM2efusion protein, which can be produced in high yields in E coli fermentors, was shown to induce a robust and long-lastingprotective antibody response in mice [151].

Subunit vaccines

A subunit vaccine made of the conserved NP internal protein, a known target for cytotoxic T cells, which is covalently coupledto the M2e peptide and to the ISS adjuvant, is in development at Dynavax Technologies, Berkeley, CA, USA. ISS, a TLR9activator, has been shown to activate NK cell secretion of IFN-? [152] and was successfully tested as an adjuvant in theHeplisavTM hepatitis B vaccine.


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VLPs

Influenza VLPs are produced in insect cell using recombinant baculovirus vectors that express viral proteins HA, NA and M1[153] [154] . Influenza VLPs induced mucosal IgG and cellular immune responses in mice, as well as long-lived antibody-secreting cells that were detected in the bone marrow of the immunized animals [155].

A two-dose immunization regimen with influenza VLPs developed by Novavax, Rockville, MD, USA, was highly immunogenicin mice and ferrets, even at the low dose of 6µg HA [156] . In contrast, an experimental trivalent influenza vaccine made ofHA0 haemagglutinins H1, B and H3 produced in serum-free insect cell cultures using three recombinant baculoviruses (ProteinScience), required 75 µg of each of the three HAs to elicit HAI antibody responses in only 51%, 65% and 81% of vaccinerecipients, respectively [157].

Live recombinant vaccines

Other influenza vaccines, such as DNA vaccines, live recombinant vaccines based on non-replicative Ad5 [158] , modifiedvaccinia Ankara (MVA) virus [159] or Newcastle disease virus vectors [160] are still at a preclinical or early clinicaldevelopment stage.

Pandemic Influenza vaccines

No current influenza vaccine would offer protection against a pandemic triggered by an emerging avian virus strain, such asH5N1, H7N7 or H7N2. Specific 'pandemic' vaccines need therefore to be prepared for that purpose. In addition, two doses ofpandemic strain vaccine spaced by one month will likely be necessary as no humans have any underlying immunity to avianinfluenza virus strains [161] . Given the world's current vaccine production capacity, if a monovalent inactivated pandemicinfluenza vaccine were produced according to the formulation of seasonal vaccines, i.e. with 15 ?g haemagglutinin per dose, onlyabout 475 million people could be vaccinated. The WHO has been active at encouraging pandemic influenza vaccine R&D andreviewing progresses through a number of meetings. Updated results on clinical trials of pandemic influenza vaccines can befound at [162].

Inactivated split vaccines

Immunogenicity trials with candidate split inactivated vaccines prepared with a H5N1 virus strain (A/Vietnam/2004 H5N1)showed disappointingly low immune potency, as two 45 ?g haemagglutinin doses of split vaccine at four weeks interval elicitedonly 38% seroconversion among 2-9 years old children and two doses of 90 ?g H5 haemagglutinin only 35% responses in elderlyvolunteers [163] [164] [165] . The need to enhance the immunogenicity of inactivated H5N1 vaccines and to search for 'antigen-sparing' formulations was therefore obvious [78] [166] [167].

Attempts at using aluminium salts as adjuvants in inactivated H5N1 influenza vaccines had a limited effect on the potency of thevaccines, as two doses of 30µg or even 45 µg HA were required for induction of six-month antibody persistence [168] . Incontrast, remarkable results were obtained with adjuvants such as polyoxidonium (Microgen), MF59 (Novartis), AS03 (GSK) orAF03 (Sanofi Pasteur), which are based on oil-in-water emulsions. These adjuvants considerably increased the immunogenicityof the inactivated split H5N1 vaccines, allowing a significant reduction in the dose of haemagglutinin needed to elicit protection.

Thus, two doses of MF59-adjuvanted Novartis vaccine (FluadTM) containing 7.5 ?g of H5 haemagglutinin elicited a protectiveinfluenza neutralizing antibody response in 77% of recipients [169] ; the antibody response to two doses of vaccine containing15µg HA (H5N1) with MF59 were higher than the response to 45 µg of vaccine alone [170] . Quite similarly, two doses ofAS03-adjuvanted GSK H5N1 vaccine containing 3.8 ?g and 7.5 ?g H5 haemagglutinin elicited haemagglutination-inhibiting(HAI) antibodies in 82% and 90% of the volunteers, as compared to 4% and 16% with the non adjuvanted vaccine controls,respectively [171] [172] ; and two doses of AF03-adjuvanted H5N1 Sanofi Pasteur vaccine containing 1.9 ?g or 3.75 ?g ofantigen generated a high level seroprotective immune response in over 70% and over 80% of the participants, respectively [44] .In addition, the antibodies elicited by the adjuvanted vaccines were broadly cross-neutralizing antibodies that neutralized virusstrains belonging to the various H5N1 virus clades in cell culture and could protect ferrets from challenge with virus strains fromdifferent H5N1 clades [173].

There is therefore no more theoretical impossibility to manufacture 1.5-2 billion doses of split pandemic influenza vaccine usingthe currently available manufacture facilities if need be. Further improvements aimed at up-scaling vaccine production in case ofa pandemic would be to develop cell-culture vaccines, which would bypass the bottleneck of limited egg supply. Influenza virus


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can be adapted to grow in a variety of cell lines including Vero cells and PER.C-6 cells, which would allow large-scaleproduction of the virus.

The possibility is also entertained that the addition of adjuvants to seasonal influenza vaccines might be of benefit to populationsat risk such as persons with underlying chronic diseases [174] . Inactivated whole-virion vaccines adjuvanted with aluminiumhydroxide were found to have better immunogenicity in naive individuals than split vaccines, but may be associated with febrilereactions, particularly in childen [175] . New adjuvants based on TLR ligands are been developed that could eventually be usedfor influenza vaccines, such as the deoxynucleotide IC31TM (Intercell, Austria) [176] or the Salmonella typhimurium flagellintype 2 (STF2, VaxInnate Corp, USA).

Inactivated whole-virion vaccines

Candidate pandemic inactivated whole-virion vaccines also have been produced by several companies, including Baxter, Austria,Nobilon International, The Netherlands, Sinovac, China, and Omnivest, Hungary. Adjuvantation with aluminium hydroxide wasfound to be devoid of effect on the immunogenicity of the Vero cell-derived Baxter H5N1 vaccine in mice and humans [177] ,whereas it provided high immunogenicity to the Nobilon vaccine in ferrets. The Omnivest H5N1 vaccine (FluvalTM), anegg-based whole-virion vaccine adjuvanted with aluminium phosphate, was found to elicit significant protective cross-cladeimmune responses in 70% of volunteers, including 60-90 years-old persons and children, after only a single IM dose of 6µg HA[178] . The Baxter vaccine [177] and the Sinovac vaccine [175] both necessitated two doses of vaccine with somewhat higherantigenic content 3-4 weeks apart to elicit protective neutralizing antibody levels in volunteers (For review, see [45]).

LAIVs

Candidate pandemic LAIVs have also been prepared. A single-dose immunization of ferrets with a candidate Flumist LAIVprepared from the A/Hong Kong/97 (H5N1) strain provided 100% protection against challenge of the animals with a variety ofH5N1 strains belonging to different clades. A series of LAIVs were produced by MedImmune (USA) by reassortment betweenavian influenza A viruses H5N1, H7N3 or H9N2 and the ca Ann Arbor virus strain (H1N1). Phase I clinical trials of resultingca reassortants were performed on volunteers kept in an isolation unit at Johns Hopkins Medical Center, Baltimore, MD, USA.Virus shedding into nasal secretions was detected for one day in 60% of volunteers and for up to four days in another 25%. Aftera second immunization a few weeks later, no viral shedding could be detected. A systematic comparison between avian-humanand human-human ca reassortants in human volunteers however evidenced reduced infectivity and immunogenicity of theavian-human LAIVs, as compared with human-human H1N1 or H3N2 LAIVs. The H5 LAIV was the least immunogenic of thefive ca reassortants tested (for a review, see [45]). A two-dose immunization with the H5N1/influenza A/Ann Arbor 60 ca(H3N2) reassortant LAIV fully protected mice and ferrets against pulmonary replication of homologous and heterologouswild-type H5N1 viruses [179].

Another avian-human ca reassortant influenza virus strain was developed as LAIV by Microgen, Russia [180] , that elicitedstrong cross-clade protection in mice. The resulting LAIV strain, which carries a H5N2 antigenicity, was tested in Phase I andPhase II clinical trials using two immunizations 21 days apart and showed reasonably good immunogenicity.

Still another type of H5 LAIV was developed by Green Hills Biotech, Vienna, Austria, by deleting the NS1 viral gene, avirulence factor known to antagonize interferon, from a H5N1 avian strain. The resulting ?NS1 H5N1 strain can only be grownin Vero cells, shows attenuation in mice and ferrets and provides protection of the animals against virulent challenge withwild-type H5N1 virus strains [181].

VLPs

A H5N1 VLP containing the HA, NA and M1 viral proteins was developed at Novavax, USA, and found to elicit strong HAIand cell-mediated immune responses in mice and ferrets and to protect 100% of the animals against cross-clade H5N1 viruschallenge. A two-dose Phase I trial is planned to take place shortly. A new recombinant VLP candidate vaccine was recentlydeveloped by LigoCyte Pharmaceuticals in collaboration with the Batelle Biomedical Resaearch Center based on the expressionof a fusion protein between the murine leukemia virus Gag protein and the influenzavirus HA glycoprotein in a baculovirus-insect cell expression system. The resulting HA-pseudotyped Gag VLPs were purified as 100 nm spherical particles that elicitedrobust anti-influenza immunity in ferrets and protected the animals against cross-clade H5N1 virus challenge [182].

Live vectored vaccines


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Live recombinant H5N1 vaccines also have been developed but still are at preclinical development stage. These include amixture of three Ad5 recombinants that expressed the HA, M1 and NP proteins from a H5N1 strain, respectively; the vaccineelicited strong protection against lethal H5N1 challenge in mice and chickens [183].

An MVA recombinant that expressed the H5 HA protein elicited protection against challenge with three antigenically distinctstrains of H5N1 influenza viruses in mice [184] . Another MVA recombinant which will express all five M1, M2, HA, NA andNP proteins is being developed and should be tested shortly.

Last, but not least, a NDV recombinant that expresses the H5 HA protein was found to be highly immunogenic and protective inchickens and could be used as bivalent vaccine against both Newcastle disease and highly pathogenic H5N1 avian influenza virusinfection in poultry [160] [185].

Subunit vaccines

A subunit recombinant H5 vaccine based on HA protein expressed in a baculovirus expression system was well tolerated inhuman volunteers but showed mediocre immunogenicity [186] , probably due to lack of oligomerization of the antigen [187].

'Pre-pandemic' vaccines.

Many plans have been made on how to mitigate an influenza pandemic once it breaks out, including stockpiling of antiviral drugs,and restricting population movements until a vaccine is available for the specific strain that has broken out. As strain-specificvaccines would be available only several months into the pandemic and would be in short supply, the possibility has beenentertained of using 'pre-pandemic' vaccines which would not match the exact pandemic strain but an earlier variant. As seenabove, H5N1 vaccines do show cross-clade protection in animal models and thus might confer sufficient protection against deathor severe disease due to a new emerging pandemic H5N1 variant. The World Health Organization is planning to stockpile morethan 100 million doses of pre-pandemic H5N1 vaccines and several nations are considering the same.

Respiratory syncytial virus and parainfluenza viruses

- Introduction- Disease Burden- Virology- Vaccine

Introduction

Respiratory syncytial virus (RSV) and parainfluenza viruses (PIV) belong to the same family Paramyxoviridae. Theirimportance as respiratory pathogens in young children has been recognized for over 40 years, yet the development of vaccinesagainst RSV or PIV has been hampered by several factors, including the risk of potentiation of naturally occurring disease, aswas observed in the early 1960s (see below).

Respiratory syncytial virus (RSV) and parainfluenza viruses (PIV) belong to the same family Paramyxoviridae. Theirimportance as respiratory pathogens in young children has been recognized for over 40 years, yet the development of vaccinesagainst RSV or PIV has been hampered by several factors, including the risk of potentiation of naturally occurring disease, aswas observed in the early 1960s (see below). RSV is the most important cause of viral lower respiratory tract illness (LRI) ininfants and children worldwide [16] , being responsible for 70 000 to 126 000 infant hospitalizations for pneumonia orbronchiolitis every year in the USA alone. The elderly also are at risk for severe RSV disease [19] [188] and 14 000 to 62 000RSV-associated hospitalizations of the elderly occur annually in the USA [189].

Human parainfluenza viruses types 1, 2 and 3 (PIV1, PIV2 and PIV3, respectively) are second only to RSV as important causesof viral LRI in young children [10] , being recovered from 18% young children [190] , with upper respiratory illness (URI), 22%with LRI and 64% with croup [191] . PIV-1 and PIV-2 are the principal causes of croup, which occurs mostly in children 6 to 48months of age, whereas PIV-3 causes bronchiolitis and pneumonia predominantly in children less than 12 months of age.

Disease Burden


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RSV infection

Human RSV infection, the single most important cause of severe respiratory illness in infants and young children and the majorcause of infantile bronchiolitis, is the most frequent cause of hospitalization of infants and young children in industrializedcountries [192] . In the USA alone, from 85 000 to 144 000 infants with RSV infection are hospitalized annually [17] , resultingin 20%-25% of pneumonia cases and up to 70% of bronchiolitis cases in the hospital [193] [194] . Global RSV disease burden isestimated at 64 million cases and 160 000 deaths every year.

RSV disease spectrum actually includes a wide array of symptoms, from rhinitis and otitis media to pneumonia and bronchiolitis.Humans are the only known reservoir for RSV. Spread of the virus from contaminated nasal secretions occurs via largerespiratory droplets, and close contact with an infected individual or contaminated surface is required for transmission. The viruscan persist for several hours on toys or other objects, which explains the high rate of nosocomial RSV infections, particularly inpaediatric wards. In the USA, nearly all children by 24 months of age have been infected at least once with RSV, and about halfhave experienced two infections [195].

Children who experience RSV infection early in life run a high risk of subsequent recurrent wheezing and asthma [196] [197][198] , especially premature infants and infants with bronchopulmonary dysplasia, for whom preventive passive immunizationwith anti-RSV monoclonal antibodies such as Palivizumab is highly recommended [199] [200] [201] . RSV-infected infant seraand nasal secretions show a marked increase in levels of Th-2 cytokines and chemokines, including IL-4 and MIP-1?, as well asof non-neutralizing IgE antibodies. The RSV G protein is believed to induce the release of large amounts of Th-2 cytokines andMIP-1? from CD4+ T cells and mast cells, basophils and monocytes, that trigger increased pulmonary eosinophilia and asthmaexacerbation [202] . Repeated RSV infection in a mouse model similarly induces persistent airway inflammation andhyperresponsiveness which are characteristics of asthma [203] [204] [205].

Infants who had been immunized with a formalin-inactivated RSV vaccine in the 1960s similarly experienced enhanced RSVdisease and pulmonary eosinophilia upon subsequent RSV infection, leading to numerous hospitalizations and two deaths [206][207] [208] , probably due to skewing of the immune response towards a Th-2 response and failure of the vaccine to induce aCD8+ T cell response [209] [210].

RSV also is a significant problem in the elderly [188] , in persons with cardiopulmonary diseases [211] and inimmunocompromized individuals [212] . RSV attack rates in nursing homes in the USA are approximately 5%-10% per yearwith a 2%-8% case fatality rate, amounting to approximately 10 000 deaths per year among persons >64 years of age [213] ].Among elderly persons followed for 3 consecutive winters, RSV infection accounted for 10.6% of hospitalizations forpneumonia, 11.4% of hospitalizations for obstructive pulmonary disease, 5.4% for congestive heart failure and 7.2% for asthma[214].

Few population-based estimates of the incidence of RSV disease in developing countries are available, although existing dataclearly indicate that the virus accounts for a high proportion of ARIs in children. Studies in Brazil, Colombia and Thailandsuggest that RSV causes 20-30% of ARI cases in children from 1-4 years of age, a proportion similar to that in industrializedcountries. Another confusing aspect of the epidemiology of RSV infection is the seasonality of the disease. In Europe and NorthAmerica, RSV disease occurs as well-defined seasonal outbreaks during the winter and spring months. Studies in developingcountries with temperate climates, such as Argentina, have shown a similar seasonal pattern. On the other hand, studies intropical countries often have reported an increase in RSV in the rainy season but this has not been a constant finding. Culturaland behavioral patterns in the community might also affect the acquisition and spread of RSV infection.

Parainfluenza virus infection

Parainfluenza viruses also cause a spectrum of respiratory illnesses, from upper respiratory infections, 30-50% of which arecomplicated by otitis media, to lower respiratory infections, about 0.3% of which require hospitalization. Most children areinfected by human parainfluenza virus type 3 (PIV-3) by the age of two years and by parainfluenza virus types 1 and 2 (PIV-1and PIV-2) by the age of five years [191] . PIV-3 infections are second only to RSV infections as a viral cause of serious ARIin young children. Pneumonia and bronchiolitis from PIV-3 infection occur primarily in the first 6-12 months of life, as is thecase for RSV infection [190] . Croup is the signature clinical manifestation of infection with other parainfluenza viruses,especially PIV-1, and is the chief cause of hospitalization from parainfluenza infections in children two to six years of age [191]. The proportions of hospitalizations associated with PIV infection vary widely in hospital-based studies. Consequently, theannual estimated rates of hospitalization fall within a broad range: PIV-1 is estimated to account for 5,800 to 28,900 annualhospitalizations in the USA, PIV-2 for 1,800 to 15,600 hospitalizations, and PIV-3 for 8,700 to 52 000 hospitalizations. Along


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with RSV, parainfluenza viruses are also leading causes of hospitalization in eldrly with community-acquired respiratorydisease.

PIV-1 causes large, well-defined outbreaks, marked by sharp biennial rises in cases of croup in the autumn of odd-numberedyears. Outbreaks of infection with PIV-2, though more erratic, usually follow type 1 outbreaks. Outbreaks of PIV-3 infectionsoccur on a yearly basis, mainly in spring and summer. Although PIV-1, -2 and -3 have been described as a cause of ARI indeveloping countries, the corresponding disease burden has not been accurately determined in these countries.

Reinfection with any of the parainfluenza viruses and/or with RSV can occur throughout life [215] , usually resulting in mildupper respiratory infections in young adults, but causing severe disease in immunocompromized patients [16] [216] [217].

Virology

RSV and parainfluenza viruses belong to the family Paramyxoviridae. These are enveloped viruses with a negative-sense single-stranded RNA genome.

Human RSV, together with its close relative bovine RSV, belongs to the subfamily Pneumovirinae, genus Pneumovirus. Itsgenome is a 15 222 nucleotide-long, negative-sense RNA molecule which encodes 11 viral proteins, among which thenucleoprotein (N), the fusion protein (F), the surface glycoprotein (G), the matrix protein (M) and several non-structuralproteins including the L protein (replicase) and virulence factors NS1 and NS2 that mediate resistance to IFN-?/? [218] . Thetight association of the RNA molecule with the viral N protein forms a nucleocapsid wrapped inside the viral envelope, fromwhich protrude viral proteins F, G and SH. The RSV G protein was shown to be a structural and functional mimetic offractalkine, a proinflammatory CX3C chemokine that mediates leucocyte migration and adhesion [219] , which explains its rolein pathogenesis [220] [221] [222].

The fusion protein F and attachment glycoprotein G are the only two components that induce RSV neutralizing antibodies. Thesequence of the F protein is highly conserved among RSV isolates. In contrast, that of the G protein is relatively variable [223] :two serogroups of RSV strains have been described, the A and B groups, based on differences in the antigenicity of the Gglycoprotein. Current efforts are directed towards the development of a vaccine that will incorporate strains in both groups, orwill be directed against the conserved F protein (for a review, see [224]).

Parainfluenza viruses belong to the subfamily Paramyxovirinae, itself subdivided into three genera: Respirovirus (PIV-1, PIV-3,and Sendai virus (SeV)), Rubulavirus (PIV-2, PIV-4 and mumps virus) and Morbillivirus (measles virus, rinderpest virus andcanine distemper virus (CDV)). Their genome, a ~15 500 nucleotide-long negative-sense RNA molecule, encodes two envelopeglycoproteins, the haemagglutinin-neuraminidase (HN), and the fusion protein (F), itself cleaved into F1 and F2 subunits, amatrix protein (M), a nucleocapsid protein (N) and several nonstructural proteins including the viral replicase (L) [225] . Allparainfluenza viruses except PIV-1 express a non-structural V protein that blocks IFN signalling in the infected cell and actstherefore as a virulence factor [226].

Vaccine

General considerations

Development of vaccines to prevent RSV infection have been complicated by the fact that host immune responses appear to playa significant role in the pathogenesis of the disease. Early attempts at vaccinating children in the 1960s with a formalin-inactivated RSV vaccine showed that vaccinated children suffered from more severe disease on subsequent exposure to the virusas compared to unvaccinated controls (see above). These early trials resulted in the hospitalization of 80% of vaccinees and twodeaths [206] [207] [208] . The enhanced severity of disease has been reproduced in animal models and is thought to result frominadequate levels of serum-neutralizing antibodies, lack of a cellular immune response, and excessive induction of a Th2 immuneresponse with pulmonary eosinophilia and increased production of IL-4, IL-5 and MIP-1? [209] [210].

In addition, naturally acquired immunity to RSV is neither complete nor durable and recurrent infections occur frequently duringthe first three years of life. Older children and adults, however, usually are protected against severe RSV disease, suggestingthat protection does develop after primary infection.

Passive immunization in the form of RSV-neutralizing immune globulin or humanized monoclonal antibodies givenprophylactically has been shown to prevent RSV infection in newborns with underlying cardiopulmonary disease, and in small,premature infants [199] [200] [201] .This demonstrates that humoral antibody plays a major role in protection against infection.


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In general, secretory IgAs and serum antibodies appear to protect against infection of the upper and lower respiratory tracts,respectively, while T-cell immunity targeted to internal viral proteins appears to help terminate viral infections.

Although live attenuated RSV vaccines seem preferable for immunization of naive infants, nonreplicative vaccines may beuseful for immunization of the elderly and older, high-risk children, as well as for maternal immunization. In addition, RSVvaccines should induce a balanced Th1-Th2 response and cover the two antigenically distinct serogroups RSVA and RSVB.

Subunit RSV vaccines

Three types of RSV subunit vaccines have been evaluated in clinical trials [227].

Candidate vaccines based on purified F protein (PFP-1, PFP-2 and PFP-3) prepared from RSV-infected cells were tested in avariety of rodent and nonhuman primate models and found to induce protection against RSV challenge [228] . These candidateswere tested in human clinical trials involving elderly volunteers [229] [230] , pregnant women [231] and children with chroniclung disease [232] [233] . The vaccines were found to be safe and moderately immunogenic but the incidence of lower RSV ARIwas not significantly diminished in the vaccinees. Vaccination of women in the 30th to 40th week of pregnancy induced RSVanti-F antibodies titres that were persistently fourfold higher in newborns to the vaccinated mothers than to those who hadreceived a placebo and was not followed by increase in frequency or morbidity of respiratory illnesses in the seropositive infants.

Another subunit vaccine consisting of co-purified F, G, and M proteins from RSV A was tested in healthy adult volunteers in thepresence of either alum or polyphosphazene (PCPP) as an adjuvant. Neutralizing antibody responses to RSV A and RSV B weredetected in 76-93% of the vaccinees, but waned after one year, suggesting that annual immunization with this vaccine would benecessary [234].

Still another subunit approach was investigated using the central domain of the RSV G protein, whose sequence is relativelyconserved among serogroup A and B viruses. A vaccine candidate, BBG2Na, was developed by fusing this domain (G2Na) to thealbumin-binding region (BB) of streptococcal protein G and producing the fusion protein in a bacterial expression system. Thecandidate vaccine elicited a protective immune response in animals [235] , and was moderately immunogenic in adult humanvolunteers [236] . Its clinical development had to be interrupted due to the appearance of unexpected type 3 hypersensitivity sideeffects (purpura) in a couple of immunized volunteers.

Live attenuated RSV and PIV-3 vaccines

The development of reverse genetic systems for RSV and parainfluenza viruses has provided for the generation of a number ofgenetically designed vaccine candidates that harbor mutations or deletions in an effort to attenuate virus replication withoutcompromising immunogenicity [237] . Achieving an appropriate balance between attenuation and immunogenicity has howeverbeen a major obstacle to the development of these vaccines.

A temperatrure-sensitive (ts) human PIV-3 (HPIV-3) strain, cp45, was selected after 45 passages of the virus in African greenmonkey cells at low temperature and evaluated as a intranasal vaccine in Phase I/II trials in RSV seropositive and seronegativechildren and in young infants. The HPIV-3 cp45 vaccine candidate was well tolerated and immunogenic in seronegative infantsas young as 1 month of age [238] [239] [240] and showed little risk of transmission to unvaccinated children and toddlers [241] .Rcp45 is a promising HPIV-3 candidate vaccine that is likely to soon be evaluated in efficacy trials [16].

A cold-passaged (cp) derivative of RSV still caused mild respiratory illness in young children: the strain was further attenuatedby chemical mutagenesis to produce the cpts 248/404 strain, which was, however, still reactogenic in 1-2 months-old infants[242] and had to be further mutagenized to produce suitably attenuated vaccine candidate strain rAcp248/404/1030-?SH [243] .Its immunogenicity remains to be tested. Another set of engineered candidate vaccines that have a deletion of the NS2 gene incommon (rAcp248/404-?NS2) have been found to be overattenuated for children [244] . Meanwhile, a combination RSV andHPIV-3 intranasal vaccine was tested in 6-18 months-old children, using as a vaccine a mixture of the RSV cpts 248/404 and theHPIV-3 cp45 strains: both vaccines were found to be as immunogenic after simultaneous administration as after separateadministration [245].

Another live attenuated candidate PIV-3 vaccine was developed using the Kansas strain of bovine PIV-3 (BPIV-3). BPIV-3 isclosely related antigenically to HPIV-3, it can protect monkeys against challenge with HPIV-3, and it replicates poorly inhumans, making a perfect Jennerian vaccine candidate. BPIV-3 was well tolerated and immunogenic in seronegative childrenand infants as young as 2 months old [246] [247] but the magnitude of the anti-HN response was lower in children who receivedthe BPIV-3 vaccine than after immunization with the HPIV-3 cp45 strain.


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Live chimeric and recombinant vaccines

A chimeric bovine/human PIV-3 (B/HPIV-3) strain was engineered by substituting in a BPIV-3 genome the HPIV-3 F and HNgenes and the F/NH intergenic sequences to their bovine equivalent. The resulting B/H chimeric virus retained the attenuatedphenotype of BPIV-3 and was highly immunogenic in rhesus monkeys [248].

The B/HPIV-3 chimeric strain was then used as a vector to express the F, or F and G open reading frames of RSV subgroup Aor B [249] , thus providing a candidate intranasal vaccine against both RSV and PIV-3 infections [250] . African green monkeysimmunized with the B/HPIV-3 chimera expressing either the native or soluble RSV F protein produced RSV-neutralizingantibodies and were fully protected against challenge with wild-type RSV [251] . The live attenuated nasal RSV/PIV-3candidate vaccine (MEDI 534TM) was shown to be safe and well tolerated in Phase I clinical studies conducted by MedImmunein the USA in adults and seropositive children 1-9 years of age. The vaccine is presently entering Phase I/IIa clinical trials in 2month-old infants and in 6-24 month-old children. The PIV-3-vectored RSV candidate vaccine could be a vaccine of choice toprevent RSV and PIV-3 infections in young infants.

Other virus vectors have been used to deliver RSV F and/or G proteins. Recombinant vaccinia virus and adenoviruses expressingRSV F, RSV G or RSV F and G were constructed and tested in animal models including chimpanzees but showed mediocreimmunogenicity [252] [253] . Sendai virus (SeV), the murine PIV-1, which had been shown to be safe in human volunteers andto protect African green monkeys against human PIV-1 challenge, was also used as a vector to express RSV fusion protein F.The recombinant SeV-RSV F induced RSV-neutralizing antibodies and RSV-specific CTLs and protected cotton rats and miceagainst challenge with RSV of both A and B subgroups [254] [255] . Similarly, a SeV recombinant expressing thehaemagglutinin-neuraminidase (HN) gene from HPIV-3 induced protection against both PIV-1 and PIV-3 challenge [256] .Sendai virus, however, does not seem to be sufficiently attenuated to be used as a Jennerian vaccine in human infants.

Venezuelan equine encephalitis virus (VEEV) replicon particles (VRPs) expressing RSV F or G similarly induced RSV-specificT cell and neutralizing antibody responses and protected mice and cotton rats against RSV challenge [257] [258].

Streptococcus pneumoniae

- Introduction- Disease Burden- Bacteriology- Vaccine- Other links

Introduction

Streptococcus pneumoniae, or pneumococcus, is a leading cause of morbidity and mortality among children worldwide andparticularly in developing countries [259] [260] [261] . It was estimated that 10.6 million children less than 5 years present withpneumococcal disease every year [262] . By far the most common form of the disease is bacteremic pneumococcal pneumonia,whose highest incidence is associated with both extremes of age (children < 2 years of age and adults >65 years of age), the nextmost common form being pneumococcal meningitis, especially in infants and young children, followed in order of decreasingincidence by blood stream infection (or sepsis) and otitis media. Sinusitis and, more rarely, endocarditis and peritonitis also havebeen reported. In developing countries, the bacterium is the leading bacterial cause of childhood ARI mortality and the leadingcause of nonepidemic childhood meningitis [263] [264].

The capsular polysaccharides (PS) on the surface of S pneumoniae, which are its primary factor of virulence, also are the basisfor the serotyping classification of the bacterium among 40 serogroups comprising 90 serotypes [265] , only 20 of which areresponsible for 70% of invasive pneumococcal disease. The most common serogroups worldwide are 6, 14, 19 and 23 [266] , butother serogroups such as 1, 5 or 8, contribute much to invasive pneumococcal disease in young children in developing countries.A matter of concern is the increasing antibiotic resistance of S pneumoniae, both in older individuals, where it accounts for anincreasing proportion of pneumococcal infections and in children less than 2 years of age, where it has added to the urgency ofdeveloping more effective pneumococcal vaccines for this age group [5].

Disease Burden

Although all age groups may be affected, the highest rate of pneumococcal disease occurs in young children and in the elderly


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population. In addition, persons suffering from a wide range of chronic conditions and immune deficiencies are at increased risk.

The only natural reservoir of S pneumoniae is the human nasopharynx, from which it can be transmitted through respiratorydroplets to other individuals. Virtually every child in the world is colonized with one or more strains of S pneumoniae andbecomes a carrier during his first years of life. In most cases carriage is asymptomatic; disease occurs in only a minority ofpersons, the bacteria spreading locally from the rhinopharynx into the sinuses and middle ear cavity or to the lungs, or causingsystemic infections including bacteremia and meningitis. Infection of the blood stream and subsequent infection of secondarysites is referred to as invasive pneumococcal disease.

In some developing countries, as for example Southern India, 50% of infants have been colonized by S pneumoniae by 2 monthsof age and 80% are carriers by the age of 6 months [267] . A study in South Africa showed that the prevalence of carriage was30%, 44%, 51% and 61% in children aged 6 weeks, 10 weeks, 14 weeks and 9 months, respectively [268] . In industrializedcountries, carriage occurs on the average at about six months of age.

Pneumococcal disease is estimated to cause more than one third of the 2 million global annual child deaths following ARIs [4] .In industrialized countries, the highest levels of infection occur in children less than 2 years of age, being the highest in thesecond 6 months of life. Thus, prior to the introduction of the conjugate pneumococcal vaccine, the annual average incidence ofinvasive pneumococcal disease in the USA was 167 cases per 100,000 population in children less than 12 months of age, with apeak at 235 cases per 100,000 among children 6 to 11 months old, and 203 per 100,000 population among children 12 to 23 monthsold. Children from minority groups suffered disproportionately, with annual incidence figures in children less than 2 years of ageof 400, 642 and 2396 per 100,000 among the black, Alaskan Native and Native American populations, respectively [261] .Reported incidence figures are lower in Europe, ranging from 14 cases per 100,000 in Germany and The Netherlands to morethan 90 per 100,000 in Spain. S pneumoniae is recognized as the first cause of infant and young children mortality, the leadingcause of meningitis and the first cause of bacteremia in less than 2 years old children in France [269] , where incidence ofpneumonia is estimated at 100,000 cases per year, causing 3500-11,000 deaths, essentially in the elderly, and that of otitis mediaat 200,000 cases per year [270].

The true burden of pneumococcal infections in children less than 5 years old is much less documented in developing countries,where disease surveillance systems and diagnostic facilities are lacking and children with invasive infections are diagnosed onlyif hospitalized. In several surveys done in sub-Saharan Africa, S pneumoniae was found to account for about 25% to >30% of thecases of meningitis in less than 5 years old children, with a case fatality rate of more than 50% [271] . Conservative estimateshave put the incidence of invasive pneumococcal disease in The Gambia at 500 per 100,000 in children in their first year of lifeand 250 per 100,000 in children less than 5 years of age [272] . A recent study in Kenya reported an annual incidence ofpresentation to the hospital with pneumococcal bacteremia of 597 per 100,000 children younger than 5 years of age [273] . Casefatality rates for invasive pneumococcal disease range from 5% to 20% for bacteremia and from 40% to >50% for meningitis.From 25% to 56% of children who survive meningitis suffer from long-term neurologic sequelae [274].

Less severe but more frequent forms of pneumococcal disease include middle-ear infection, sinusitis or recurrent bronchitis.Thus, in the USA alone, seven million cases of otitis media are attributed to pneumococci each year.

Among adults in industrialized countries, pneumococcal pneumonia still accounts for at least 30% of all cases of community-acquired pneumonia admitted to the hospital, with a case fatality rate of 11% to 44%. Annual incidence of commmunity-acquiredpneumonia in 2002 in the USA was 18.3 cases per 100,000 elderly persons, and the incidence of pneumococcal pneumonia amongthe elderly population was at least 5.5 cases per 100,000 population [275] . Again, substantially higher incidence rates werereported in blacks than in whites, and even higher rates in native Americans and Alaskan natives [276] . Similarly, a very highincidence rate was reported in the aboriginal Australian population [277] . S pneumoniae also is an underappreciated cause ofnosocomial pneumonia in hospital wards and intensive care units as well as in nursing homes and long-term care institutions.Important risk factors are age, chronic heart and lung disease, cigarette smoking, and asplenia [278].

The burden of invasive pneumococcal infections in adults in developing countries is poorly known, mostly due to failure to obtainblood cultures from patients with pneumonia. S pneumoniae has been the leading nonmycobacterial cause of pneumonia amongHIV-infected persons both in developed and developing countries [11] [279] [280] . The impact of HIV infection onpneumococcal disease can clearly be evidenced from the clinical study in HIV-infected young adults in Uganda, which reportedan annual incidence of 1700 cases of invasive pneumococcal disease per 100,000 population [281] , as well as from an earliersurvey among HIV-positive commercial sex workers in Kenya, where annual incidence of pneumococcal disease was found tobe 4250 per 100,000 people [282] . HIV-infected children seem to be 20-40 times more likely to contract pneumococcal diseasethat uninfected children.


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Influenza also increases the risk of secondary pneumococcal infection. Based on evidence from past influenza pandemics, theattack rate for secondary pneumococcal pneumonia in a pandemic setting is anticipated to reach 13% [49].

Bacteriology

S. pneumoniae is a Gram-positive encapsulated diplococcus. The external capsular polysaccharide (PS) of the bacterium is theprimary factor of virulence, other virulence factors being pneumolysin, which leads to pore formation and osmotic lysis ofepithelial cells, autolysin, and pneumococcal surface protein A (PspA), which interferes with phagocytosis and immune functionin the host.

Pneumococci are transmitted by direct contact with respiratory secretions from patients or healthy carriers. Although transientnasopharyngeal colonization rather than disease is the normal outcome of exposure to pneumococci, bacterial spread to thesinuses or the middle ear, or bacteraemia following penetration of the mucosal layer, may occur in persons susceptible to theinvolved serotype. Pneumococcal resistance to essential anti-microbials such as penicillins, cephalosporins and macrolides is aserious and rapidly increasing problem worldwide.

Capsular PS also are the basis for the serotyping classification of the bacterium among 91 serotypes. The distribution of disease-causing serotypes varies between geographic regions and by age and disease within regions. The most common serogroupsworldwide are 6, 14, 19 and 23 but some serogroups, such as 1, 5 or 8, contribute much to invasive pneumococcal disease inyoung children in developing countries. Approximately 90% of the most frequent isolates belong to 23 serogroups or serotypesand have been included in the 23-valent pneumococcal vaccine.

Vaccines

Current S pneumoniae vaccines are based on the use of the bacterial capsular polysaccharides (PS), which induce type-specificantibodies that activate and fix complement and promote bacterial opsonization and phagocytosis [11] [261] . The two types ofcurrently licensed vaccines [283] are the pneumococcal polysaccharide vaccine (PPV), based on purified capsular PS, andpneumococcal conjugate vaccines (PCV), obtained by chemical conjugation of the capsular PS to a protein carrier [284].

23-valent polysaccharide vaccine (PPV23)

The PPV23 vaccine contains 25 ?g of the purified capsular PS from each of the 23 different S pneumoniae serotypes thattogether account for 90% of cases of severe pneumococcal disease in industrialized countries. Two vaccines are currentlymanufactured, Pneumovax 23TM by Merck and Pneumo 23TM by Sanofipasteur. Relatively good antibody responses are elicitedfollowing a single IM injection in 60-80% of healthy adults and normal children over two years of age. Pneumococcalvaccination of children 2 to 5 years of age was 62% effective in preventing invasive pneumococcal disease due to vaccineserotypes [285].

In a case control study in Connecticut, the effectiveness of pneumococcal vaccination with PPV23 in immunocompetent adultswas estimated to be 61% against pneumococcal bacteremia, but in immunocompromised patients this figure fell to only 21%[286] . In spite of such suboptimal immunogenicity, administration of a single dose of PPV23 continues to be recommended forsolid-organ transplant recipients [287] . The PPV23 vaccine also is recommended for people over 65 years of age, particularlythose living in institutions. Several studies have shown that PPV23 is effective in preventing invasive pneumococcal disease [288], but it remains unclear whether it has a significant protective effect against pneumonia [289, 290]. A recent meta-analysisconcluded that there actually was little evidence of PS vaccine protection against pneumonia among elderly or adults withchronic illness [291] .

In a case control study in Connecticut, the effectiveness of pneumococcal vaccination with PPV23 in immunocompetent adultswas estimated to be 61% against pneumococcal bacteremia, but in immunocompromised patients this figure fell to only 21%[286] . In spite of such suboptimal immunogenicity, administration of a single dose of PPV23 continues to be recommended forsolid-organ transplant recipients [287] . The PPV23 vaccine also is recommended for people over 65 years of age, particularlythose living in institutions. Several studies have shown that PPV23 is effective in preventing invasive pneumococcal disease [288], but it remains unclear whether it has a significant protective effect against pneumonia [289] [290] . A recent meta-analysisconcluded that there actually was little evidence of PS vaccine protection against pneumonia among elderly or adults withchronic illness [291] . Also, PPV23 is unable to elicit immune memory, so that a second dose of vaccine does not boost antibodylevels. PPV23 does not provide protection against mucosal infection, and is thus unable to reduce nasopharyngeal carriage ofpneumococci. Moreover, studies of PPV23 in adults and children have shown that a state of immune tolerance, or


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hyporesponsiveness, can develop to repeated PS vaccine exposures [292] . Last, but not least, PPV is poorly immunogenic in lessthan 2 years old children and is thus inadapted to infants and young children.

On another hand, a number of studies have confirmed the safety of PPV during pregnancy and documented the use of the vaccinefor the vaccination of pregnant or breast-feeding mothers for preventing pneumococcal pneumonia in young infants [293] [294].

Pneumococcal conjugate vaccines

Pneumococcal conjugate vaccines (PCVs) are based on the covalent coupling of the capsular PS from diverse S pneumoniaeserotypes to a variety of protein carriers. These vaccines elicit higher antibody levels in infants, young children, the elderly andimmunodeficient persons than the PPV23 vaccine, as well as significant immune memory resulting in an anamnestic response onsubsequent booster immunizations. Moreover, they suppress nasopharyngeal carriage of the pathogen, thus decreasing bacterialtransmission in the community and generating herd immunity. Conjugate vaccines immunization followed by PS vaccine boostingmight provide a foundation for lifelong protection against pneumococcal disease and/or maintain high levels opsonophagocyticantibody titers in elderly adults while broadening serotype coverage [295] [296].

The first PCV, PrevnarTM or PrevenarTM (Wyeth), was licensed in the USA in 2000 and recommended for routine use inchildren younger than 2 years of age, to whom it is administered in a 3 doses schedule, when possible in combination with usualroutine vaccination, followed by a booster dose at 15-18 months. Alternatively, the vaccine can be administered in a two-doseimmunization schedule at 3 and 5 months of age, followed by a booster immunization at 11-12 months of age [297] [298] . Thevaccine contains poly- or oligo-saccharides from seven S pneumoniae serotypes (4, 6B, 9V, 14, 18C, 19F and 23F), eachconjugated to genetically detoxified diphtheria toxin CRM 197.

Four large clinical trials of the 7-valent PCV7 and of a closely related, unlicensed 9-valent PCV9 in the USA, South Africa, andThe Gambia have reported vaccine efficacy of between 77% and 97% against severe invasive pneumococcal disease caused byvaccine serotypes and of 19% to 37% against radiologically confirmed pneumonia [299] [300] [301] [302] . Efficacy of thevaccine against otitis media was reported to be 57% against vaccine serotypes [303] . Introduction of PCV7 in the USA resultedin a dramatic decline in the rates of invasive pneumococcal disease among children <5 years of age, which dropped from 97cases per 100,000 population during 1998-1999 to 24 cases per 100,000 in 2005; disease caused by vaccine-type strains fell from80 cases per 100,000 population to 4.6 [304] . A significantly decreased incidence of pneumococcal otitis media and acutebacterial rhinosinusitis was also noted [305] [306] . In children not at high risk for invasive disease, the effectiveness of the full4-dose schedule vaccine against vaccine serotypes was estimated to be 91%. Substantial protection against invasivepneumococcal disease and clinical pneumonia was also noted in HIV-infected infants [307].

A significant reduction in S pneumococcus disease incidence also was seen in unvaccinated individuals as a result of herdimmunity [308] [309] . Thus, in adults 65 years old and older, invasive disease dropped by about one-third since introduction ofthe conjugate vaccine for children, and a drop of similar magnitude was seen in hospitalizations for pneumococcal bactaeremia[310] . Paradoxically, after 5 years of wide use of Prevnar for infant immunization in the USA, more cases seem to beprevented through the indirect effects of herd immunity than by vaccine-induced immunity in the vaccinees [311].

Additional Phase III clinical trials also have been performed using 9-valent and 11-valent PCVs that are not expected to reachthe market. Trial of PCV9 in South Africa on 40,000 subjects showed 83% and 65% efficacy after 2.3 years of follow-up inHIV-infected and uninfected children, respectively. The figures still were 77.8% and 38.8% after 6.16 years of follow-up [312] .In The Gambia, PCV9 reduced radiologically-confirmed pneumonia by 37% and invasive disease caused by vaccine serotypes by77% [51] . The Gambian trial of PCV9 also showed a 16% reduction in hospital admissions and deaths from any cause inchildren 3-29 months of age who received the vaccine compared with those who had not received the vaccine. PCV9 couldsuccessfully be administered to infants in a two-dose schedule at 2 and 4 months of age followed by boosting at 12 months of age[313].

In a recent cost-effectiveness analysis of PCV7, it was projected that, in the 72 GAVI-eligible countries, pneumococcalvaccination with a conjugate vaccine would prevent between 262,000 and 407,000 deaths in children aged 3-29 months, dependingon the level of vaccine coverage, and not counting herd immunity effects [314] . At a price of $5 per dose, the vaccines would bea highly cost-effective purchase. In more affluent societies, where the cost of the vaccine is definitely higher, studies ofcost-benefit ratio of large scale PCV vaccination always have been highly positive [315].

These results speak very favorably in favor of the currently available conjugate vaccine and lead to expect an even greatervaccine effectiveness in infants and young children with the eventual licensing and use of the 10-valent (GSK) and 13-valent(Wyeth) conjugate vaccines which are in Phase III clinical trials at this time and are expected to be licensed in the coming


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years. The currently licensed 7-valent vaccine, PrevnarTM, does not contain some of the serotypes that cause severe disease indeveloping countries, notably serotypes 1 and 5 [316] . Serotype 1 is the most common among children over 2 years of age inmany countries in Asia and Africa [317] . Serotypes 1 and 5 are predominant In Nepal, whereas serotypes 14 and 19 arepredominant in Sri Lanka [318] . The new conjugate vaccines will provide more optimal serotype coverage in these countries.The protein carrier used by Wyeth is CRM197, a genetically detoxified mutant of diphtheria toxin, whereas that used by GSK isthe H. influenzae protein D [319] [320].

The success of PCV7 has partly been offset by the observation that introduction of PCV can lead to replacement of the vaccineserotypes by other, nonvaccine S pneumoniae serotypes. Carriage of non-vaccine type strains increased among children receivingthe PrevnarTM vaccine such that the overall prevalence of pneumococcal carriage was not different in vaccinated andunvaccinated children, new serotypes taking up the mucosal territory vacated by the pneumococcal serotypes included in thevaccine [268] [321] [322] . So far, strain replacement has only had relatively modest effects on disease, except in certainsettings [323] . Replacement disease was nevertheless observed in the otitis media trial in Finland, where the vaccine group had33% more episodes of otitis media caused by serotypes not included in the vaccine [303] . Of positive note was the significantreduction in the disparity in disease rates between black and white children following PCV introduction in the USA [324] .Serotype replacement makes it essential that there be continued monitoring and surveillance of pneumococcal colonization andinvasive disease [325].

Protein vaccines

Newer vaccine approaches are been developed, based on the use of conserved external surface proteins such as PspA and PspC,which have a choline-binding function, pneumococcal surface adhesin A (PsaA), a metal-binding transporter, surface proteinsPiaA and PiuA, or virulence factors such as pneumolysin or autolysin. Some of these antigens have been identified by a reversevaccinology approach, including the pneumococcal pilus, a potent immunogen that elicits cross-protection against various Spneumococcus serotypes [326] [327] . These new vaccines would circumvent the complexity of manufacture of conjugatevaccines and be serotype-independent [328] . Several of these candidate vaccines induced protection against systemic challengein animal models [329] . PspA and PsaA have been tested in Phase I trials and found to elicit protective anti-S pneumoniaeantibodies [330] [331] [332].

Two other surface proteins, BVH 3 and BVH 11, have been identified that can elicit protective anti-pneumococcal antibodies inthe mouse model, and a recombinant 100 kD fusion protein, BVH3/11V, was tested by ID BioMedical in a Phase I trial intoddlers and elderly volunteers. A 2-dose immunization regimen was able to induce a 50-fold increase in anti-S. pneumoniaeantibody levels. Phase II clinical studies in infants and elderly persons have been initiated. The advantage of this approach is thatthe BVH proteins seem to be conserved among the 90 serotypes of S pneumoniae and therefore could constitute a universalpneumococcal vaccine [333].

Useful Links

Tuberculosis

- Introduction- Disease Burden- Bacteriology- Vaccine- Other links

Introduction

An estimated one third of the world population (two billion people) is exposed to the risk of tuberculosis (TB), whose maincausative agent, Mycobacterium tuberculosis (Mtb), infects approximately 9 million new individuals and causes 1.7 milliondeaths every year [334] [335] . This problem is compounded by the global emergence of multi drug-resistant (MDR) Mtb strains[336] , the evidence that diabetes predisposes people to TB infection, and the increased susceptibility of HIV-infected individualsto TB, which all make the need for improved TB control even more urgent.

The currently available TB vaccine is bacillus Calmette-Guérin (BCG), a M bovis derivative which is routinely used in childrenbecause it is effective at protecting them against severe extrapulmonary forms of the disease, such as TB meningitis. BCG,which also elicits protection against leprosy [337] , does not however appear able to provide protection against pulmonary TB in


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adults [338] [339] . BCG vaccination may provide protection only against primary infection but has a limited effect onreactivation TB in already infected individuals or on TB re-infection in adults. It also has been shown that the "take" of BCGmay be impaired due to previous exposure to environmental mycobacteria [340] such as M avium [341] , which are frequent intropical countries. There is, therefore, an urgent need to develop better or improved TB vaccines, but it would be unethical andimpractical to test new vaccine strategies that would not include BCG in early infancy [342] [343] . Therefore, most new TBvaccine development is being done in a BCG prime-vaccine boost strategy.

Disease Burden

The global death toll of TB was 1.7 million deaths in 2006, 200 000 of which were from HIV-associated TB [344].

M tb currently infects about 2 billion people worldwide and causes an estimated 8.8 million new cases every year, especially inthe sub-Saharan African continent, in Southeast Asia and in Eastern Europe [345] . In view of underreporting and lack ofsystematic surveillance, the real incidence of TB is suspected to actually be perhaps as high as 14 million new TB cases eachyear. About one half of the new cases occur in China, India, Pakistan, Bangladesh, Indonesia and The Philippines. In Africa, thesingle most important factor determining the increased incidence of TB in the last 10 years is HIV infection: in some regions, upto 75% of new TB cases are in HIV-infected people [346] . Paradoxically, TB seems to be severely aggravated in these duallyinfected patients when active antiretroviral therapy is initiated.

In addition, about 500 000 new cases of multiple-drug resistant TB (MDR-TB) are reported each year worldwide, with highincidence in parts of Russia, Latvia and Estonia (up to 10% of new TB cases) and Azerbaidjan (22% of new cases). The newlydiscovered extensively drug-resistant Mtb strains (XDR-TB) emerged in 2005 in KwaZulu Natal (South Africa) among HIV-TBpatients, probably as a consequence of lack of observance of therapy [347] [348] [349] . XDR-TB strains are now been found in>45 different countries, mostly in HIV-infected patients [350] . XDR-TB is resistant to almost all drugs used to treat TB,including isoniazid, rifampicin, fluoroquinolones and amikacin, kanamycin or capreomycin [351].

TB is highly contagious. Left untreated, each patient with active TB will infect on average between 10 and 15 people every year.TB spreads readily from person to person, due to the production of small particle droplets when a patient coughs and to the lowdose of bacilli needed to initiate infection. Transmission is common in households, schools, hospitals, prisons, crowded workplaces, refugee camps and shelters. In fact, the incidence of TB in industrialized countries, which generally increased from theearly 1980s to the early 1990s, has been decreasing ever since. As an example, in the USA, 13 767 cases were reported in 2006,a 3.2% decrease from 2005 and a 53% decrease from 1992 [352] . However, TB is a poverty-related disease: it has long beenrecognized that war, malnutrition, population displacement and crowded living and working conditions favor the spread of TBamong humans, whereas periods of improvement in societal conditions and hygiene favor its rapid decline.

The global spread of the disease is also facilitated by migrations and movements of populations: thus, in industrialized countries,about one-half of TB cases occur in foreign-born or migrant persons. The incidence of TB in 2003 in France was for example 5.6cases per 100,000 population in the native population, 31.7 per 100,000 in persons born in North African countries and 187.7 per100,000 in persons born in TB-endemic sub-Saharan African countries [353] . But the major bottleneck for higher success ratesin controlling TB is the fact that currently only about 40% of all sputum-positive TB are detected. Thus, the majority of TB casesremains untreated or is treated only at a very late and highly infectious stage, causing enormous individual hardship as well ascreating a public health time bomb. For all these reasons, added to the relative ineffectiveness of the current BCG vaccine, thedevelopment of improved TB vaccines has become a necessity for adequate control and elimination of the disease.

Bovine tuberculosis, caused by M bovis, is a zoonotic disease and was the cause of many human deaths in the 1930s and 1940sthrough consumption of contaminated milk and dairy or meat products. Compulsory eradication programs were introduced inmany countries based on the slaughter of infected cattle detected by the intradermal tuberculin skin test. However, probably dueto a wildlife reservoir, the incidence of TB in cattle has exponentially increased over the last two decades in certain countries,especially Great Britain, constituting a potential public health problem and calling for the development of more specificdiagnostic reagents [354].

Bacteriology

Mycobacterium tuberculosis (M tb), the agent of human TB, was discovered in 1882 by Robert Koch and for a long time calledafter his name (the 'Koch bacillus'). All members of the Mycobacterium genus share the property of acid-fastness (Ziehl-Neelsen staining), due to their mycolic acid-rich cell wall structure. They include M. tuberculosis, M. africanum, and M.ulcerans, which are primary human pathogens, M. bovis, the agent of TB in cattle and other animals, which also can cause


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disease in humans, and a great many nontuberculous or environmental species, some of which can be pathogenic in humans suchas those belonging to the M. avium-intracellulare complex.

TB bacilli usually multiply first in the lung alveoli and alveolar ducts and in draining lymph nodes, where they are engulfed bydendritic cells (DCs). They also multiply in the macrophages that were attracted from the bloodstream and are armed to kill thebacteria upon uptake in their phagosomes. However, Mycobacteria can evade the phagosome-lysosome fusion pathway, multiplyin the host macrophages and kill them, progressively creating a primary tubercle. The outcome of infection is controlled by CD4+and CD8+ T cells, both of which are necessary for the maintenance of a latent state of infection [355] [356] . Delayed cutaneoushypersensitivity develops and together with other cellular immune reactions, leads to the caseous necrosis of the primarycomplex. Bacilli eventually spread to many parts of the body such as liver, spleen, meninges, bones, kidneys and lymph nodes,where they can either be a source of overtly disseminated TB or, more commonly, remain dormant. Occasional decline incell-mediated immunity leads to reactivation TB, most frequently seen in adults as a pulmonary disease with infiltration or cavityin the apex of the lung. This is the most common and most infectious form of TB.

CD4+ T-cells play a major role in containment of TB infection; progressive TB is usually associated with a Th2 T-cell response,whereas a pure Th1 response, including production of IL-2 and IFN-?, mediates protection [357] . CD8+ T cells andmacrophages are involved in the control of mycobacterial infection [358] [359] . The production of Th1 associated cytokines suchas IFN?, TNF? and IL-12 appears to be an essential component of resistance to Mtb infection. The most effective vaccinationstrategies in animal models have been those that stimulate T cell responses, both CD4+ and CD8+, to produce these cytokines.

The tuberculin skin test has long been used as evidence of TB infection or as a sign of adequate response to BCG vaccination,although no clear relationship between delayed-type hypersensitivity and protective immunity could be established [346] . Anumber of antigens found in M. tuberculosis, including Ag85, MPT64, ESAT-6 and CFP10, have been identified which may playa major role in cellular immunity and the induction of a protective IFN-? response.

Vaccine

BCG

By culturing a M. bovis isolate from a cow for a period of 13 years and a total of 231 passages, Calmette, a physician, andGuérin, a veterinarian, created an attenuated variant of M. bovis, Bacille Calmette-Guérin (BCG). BCG was first tested ininfants as an oral vaccine in 1921. New methods of administration were later introduced, such as intradermal, multiple puncture,and scarification [360] . BCG vaccination was included in the WHO Expanded Programme on Immunization (EPI) as of 1974:since then, approximately 100 million children received a BCG vaccine each year, and, to date, well over 4 billion people havebeen vaccinated with BCG since the historical immunization in 1921.

However, WHO stopped recommending BCG vaccination at birth as of 2007, at least for infants at risk for HIV infection, asdata showed that about 417 per 100 000 infants developed disseminated BCG disease due to antenatal infection with HIV [361] .Disseminated BCG disease typically presents with the same symptoms as severe TB and shows a CFR of 75%-86% [362] [363].

As shown by sequencing, the original BCG strain lost the RD1 region of the M. bovis genome in the course of the selectionprocess. This region encodes major TB antigens including culture filtrate protein 10 (CFP-10) and early secretory antigen target6 (ESAT-6) [364] [365] [366] [367] . Major BCG vaccine strains in use today differ even further from the original BCG strainand from each other, with "stronger" strains (Pasteur 1173 P2, Danish 1331) being more reactogenic and, presumably, moreimmunogenic, than "weaker" strains (Glaxo 1077, Tokyo 172) [346] [368].

No other widely used vaccine is as controversial as BCG. Its effects in large randomized, controlled, and case-control studies,have been widely disparate, from excellent protection against TB to no protection. Most studies have demonstrated that BCGvaccines afford a higher degree of protection against severe forms of TB, such as meningitis and disseminated TB, than againstmoderate forms of the disease. The efficacy of neonatal BCG vaccination also wanes with age [369] . Studies that evaluatedmeningitis or miliary TB demonstrated that BCG can provide good protection against these serious forms of TB in youngchildren, with reported efficacy ranges from 46-100%. In contrast, efficacy against pulmonary TB, which is more prevalent inadolescents and adults, has ranged from 0-80%. Overall, BCG is only at best credited with a 50% efficacy [370] [371].

Efficacy of BCG vaccination also appears to vary with geographic latitude - the farther from the equator, the more efficaciousthe vaccine. Presumably, exposure to nonpathogenic mycobacteria, which is more intense in warm climates, induces a degree ofprotective immunity in exposed populations, masking potential protection from BCG.


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Vaccination with BCG nevertheless remains the standard for TB prevention because of its efficacy in preventing life-threateningforms of TB in infants and young children, and also because it is the only vaccine available, is inexpensive, and requires only oneencounter with the baby. The fact that BCG does not protect against pulmonary TB in adults has however prompted the searchfor new, improved TB vaccines. Dozens of TB vaccine candidates have been tested in recent years in animal models, includingsubunit protein and peptide vaccines, DNA vaccines, rationally attenuated Mtb strains, recombinant BCG, and live vectorsexpressing immunodominant Mtb antigens (for reviews, see [372] [373] [374] [375]).

Among the many innovative new approaches that have been tested, one has been to reengineer BCG strains to endow them withthe capacity of expressing immunodominant Mtb antigens. This approach has yielded a series of vaccine candidates, such as:

- A recombinant BCG vaccine (BCG30) that was engineered at University of California at Los Angeles (USA) to express the 30kD major secretory protein Ag85B [376] [377] . The vaccine was tested in Phase I trial in the USA and elicited markedlyenhanced central and effector memory Ag85B-specific CD4+ Th1 and CD8+ T cell immunity compared with the parental BCGTice strain and induced a significant number of Ag85B-specific T cells capable of inhibiting intracellular mycobacteria [378].The development of this vaccine is currently on hold for regulatory concerns.

- A BCG::RD1 recombinant, in which the RD1 segment of the M. tuberculosis genome has been reintroduced, resulting in theexpression of ESAT-6 and Ag85A proteins. This new BCG strain, developed at the Pasteur Institute, Paris, showed increasedpersistence and improved protection against challenge with virulent M tb in animal models [379] but meets with safety concernsfor its use in humans.

- Another recombinant BCG, VPM 1002 (previously known as rBCG: [delta] ureC-Hly), was engineered at the Max PlanckInstitute for Infection Biology in Berlin (Germany) to express listeriolysin O, which increases MHC class I presentation [380] ,and its urease gene was deleted in order to prevent neutralization of the acidic pH in phagosomes. This recombinant BCG wasfound to be devoid of pathogenicity for SCID mice and provided greatly improved protection against aerosol TB in the mousemodel. This vaccine is currently phase I clinical trials.

- AERAS-422, developed by the Aeras Global TB Vaccine Foundation, which expresses Mtb antigens 85A, 85B, and Rv3407,together with perfringolysin, a pore-forming protein similar to listeriolysin, is currently in late preclinical development andplanned to enter into Phase I trials by the end of 2010 [381].

The VPM 1002 and AERAS-422 vaccines have been found to be more efficacious than classical BCG against Mtb challenge inanimal models, including against challenge with the Beijing strain of Mtb which is relatively insusceptible to BCG [382].

Live attenuated Mtb mutants

Another approach has relied on the engineering of live attenuated mycobacterial strains, either auxotrophic mutants of Mtb [383][384] , or regulatory mutants such as the PhoP knock-out mutant [385] . PhoP is a gene which controls the expression of severalvirulence genes of Mtb. The PhoP/PhoR Mtb mutant has shown very good protection in mice and was one of the rare new TBcandidate vaccines to show significant improvement over BCG in the guinea pig aerosol model [386] . Concern that auxotrophicmutants could revert to full virulence in vivo has led to the engineering of mutants bearing two independent unlinked deletions[387].

Subunit and DNA vaccines

Due to safety concerns, in particular in immunocompromised persons, as well as to technical challenges regarding manufactureand reproducibility, live mycobacteria vaccines are not the product of choice of most vaccine manufacturers. Many new TBvaccines approaches are therefore focused on recombinant subunit vaccines, DNA vaccines [388] , or live-vector recombinantvaccines that express a variety of M tb antigens.

An analysis of the Mtb proteome led to the identification of about 45 potential vaccine antigens. Among those, purifiedmycobacterial antigens such as Ag85A, Ag85B, HSP65, the R8307 protein, a 36kD proline-rich mycobacterial antigen, or the19kD and 45kD proteins have been found to induce protection levels in mice similar to that obtained with BCG when presented incombination with Th1-inducing adjuvants [389] [390] . Subunit vaccines based on the same proteins in adjuvants used incombination with BCG have resulted in better protection against experimental challenge of cattle with M bovis than BCGvaccine on its own [391].

Most promising among these were Ag85 (A and B), and the Mtb9.8, Mtb9.9, Mtb11, Mtb32, Mtb39, Mtb41 and ESAT-6 proteins


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[392] [393] [394] [395] , which were prioritized for vaccine development based on their ability to stimulate PBMC responsesfrom more than 50% of healthy PPD+ donors who presumably have contained their infection due to protective CD4+ T cellresponses [342] [396].

Some of these proteins have been fused together, such as Mtb32 and Mtb39, yielding M72, which was found to be highlyimmunogenic and protective in monkeys when formulated in the AS02A adjuvant from GSK, or antigens 85B and ESAT-6,whose fusion product also induced strong protective immune responses in monkeys [397] [398] . ESAT-6 could advantageously bereplaced by TB10.4 [399] . M72 is being developed by Corixa Corp and GSK and was found to be safe and immunogenic inPhase I clinical trial [400].

Antigen 85A, a mycolyl-transferase that is involved in cell wall biosynthesis, is also considered a leading candidate for inclusionin a TB vaccine. In addition, a multi-epitope polypeptide, as well as nonproteinic antigens such as mycolic acids andcarbohydrate moieties, are being developed as candidate antigens.

Live recombinant TB vaccines.

Modified vaccinia virus Ankara (MVA) was engineered to express M tb Ag85A (MVA85A) and tested successfully in a BCGprime-recombinant MVA boost strategy in the mouse challenge model, the guinea pig aerosol challenge model and thecynomolgous macaque challenge model [386] , before undergoing Phase I clinical studies first in the UK, then in a high TBendemicity setting in The Gambia [343] [401] . The MVA recombinant was found to boost BCG-primed and naturally acquiredanti-mycobacterial immunity. Interestingly, the MVA85A vaccine induced up to 30-fold greater cellular immune responses inBCG-primed than in BCG-naive individuals, even if BCG had been administered as long as 38 years before the MVA boost.Another Phase I clinical trial recently took place in South Africa [402] . The BCG/MVA85A prime-boost strategy was alsotested in cattle, resulting in significantly wider Ag85A-specific T cell responses and higher frequencies of Ag85-specific IFN-?secreting cells [403] . Currently, the prime-boost strategy is being tested in a Phase IIa trial in South Africa, and a Phase IIbtrial is planned to begin in early 2009 on 4 months-old infants who had a BCG vaccination at birth. The MVA85A vaccine iscurrently the most advanced candidate TB vaccine [404].

Live, nonreplicative adenoviruses expressing M tb antigens have been developed by the Aeras Global TB Vaccine Foundation(USA) and Crucell (Netherlands). Advanced TB vaccine candidates in this category include AERAS 407, a replication-defectiveadenovirus 35 construct that expresses Mtb Ag85A, Ag85B and TB10.4, and went through Phase I trial in South Africa and SouthAmerica; and AERAS X05, a Shigella-delivered recombinant double-stranded RNA encoding Ag85A, Ag85B and Rv3407,which is expected to enter clinical trials shortly. The immunogenicity of a BCG-AdAg85A prime-boost regimen in cattle wasfound to be significantly greater than that of either vaccine separately [405].

It is clear that, from now on, human clinical studies will act as the principal driving force for the development of new TBvaccines. Testing of such a wide variety of vaccine types using different immunization strategies directed against a sole pathogenis unique in the history of vaccine development. It will make the valid comparison of clinical data most challenging [406] .Therefore, it will be all the more important that this effort be tightly coordinated to provide maximal comparability andtransparency. WHO is working with all stake holders in the field to standardize key parameters such as trial entry criteria,endpoints, immunoassays, etc. The main players in this area include the US National Institute for Allergy and InfectiousDiseases (NIAID), the Aeras Global TB Vaccine Foundation, supported by the Bill and Melinda Gates Foundation, the WelcomeTrust, a network of European researchers supported by the European Commission, the European and Developing CountriesClinical Trial Partnership, and pharmaceutical manufacturers including GlaxoSmithKline (GSK), IDRI-Corixa, Crucell,SanofiPasteur and Emergent Biosolutions. Discussions are ongoing between these major players to develop a plan for the designand funding of Phase III efficacy trials in appropriate field trial sites in Africa and Asia [375].

The A/2009 H1N1 influenza virus pandemic

IntroductionDisease Burden and EpidemiologyVirologyVaccines

Introduction

Since the global H1N1 influenza virus pandemic of 1918, influenza virus gene reassortment has been well documented and


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observed to occur frequently between human virus subtypes, between human and avian and among avian influenza viruses. Suchreassortments led to the global pandemics of 1957 (H2N2) and 1968 (H3N2) [407] [408] . Although A/H1N1 viruses continued tocirculate among humans, seasonal epidemics of influenza A virus from 1968 to 2009 were dominated by A/H3N2 virus variantsgenerated by antigenic drift [409] [410] , until, In early April 2009, a new influenza A (H1N1) virus brutally emerged amonghumans in California and in Mexico, quickly spreading worldwide through human-to-human transmission, and generating the firstinfluenza pandemic of the twenty-first century [411] [412] . The virus was found to be antigenically unrelated to human seasonalinfluenza viruses but genetically related to viruses known to circulate in pigs. In view of its likely swine origin, it is oftenreferred to as 'swine-origin influenza virus' (S-OIV) A/H1N1, or 2009 A (H1N1) influenza virus.

Swine H1N1 influenza viruses had been circulating in pig populations for at least 80 years but too often lacked surveillance andmolecular characterization. In 1998, a new triple-reassortant H3N2 virus emerged in the North American pig population,comprising genes from classical swine H1N1, North American avian and human H3N2 influenza viruses. Co-circulation andmixing of this North American triple-reassortant with viruses of swine lineage generated further H1N1 and H1N2 reassortantswine viruses. In Europe, an avian H1N1 virus ('avian-like' swine H1N1) was first isolated from pigs in Belgium in 1979 [413] ,and gradually replaced classical swine H1N1 viruses [414] . The 'avian-like' virus lineage spread all over Europe and Asia whilealso reassorting with other influenza virus strains. In Asian pig populations, the classical swine H1N1 virus lineage stillcirculated together with the 'avian-like' swine H1N1, H1N2 reassortants and the North American H3N2 triple-reassortant [415] .Multiple lineages of influenza A viruses have been found to co-circulate during any single season and to undergo frequentreassortment which may in turn have a major impact on antigenic evolution [416] .

Molecular studies of the new A/H1N1 pandemic virus genome showed that it was derived from several viruses which had beencirculating in pigs for a long time, namely the North American H3N2 triple-reassortant, the classical swine H1N1 lineage, andthe Eurasian 'avian-like' swine H1N1 virus (see details under Virology). Initial transmission to humans is believed to have takenplace at least several months before recognition of the first outbreak and phylogenetic data even suggest that the reassortment ofswine lineages may have occurred years before emergence in humans [417] [418] [419] [420] . Surprisingly however, there hasbeen no evidence so far that swine are playing any role in the epidemiology or in the worldwide spread of the virus in humanpopulations [421] .

On June 11, 2009, the World Health Organization raised the pandemic alert to level 6, in view of the number of regions whichofficially reported A/H1N1 influenza cases in their communities. In view of the rapid spread of the H1N1 virus, its propensity toprimarily affect children and young adults, as well as those with an underlying lung or cardiac disease condition [422] , and therisk of a possible increase in pathogenicity through further reassortment with avian or human virus strains, the development of aspecific vaccine was promptly engaged in collaboration between the World Health Organization, Health Ministers and NationalHealth Agencies and the vaccine industry.

Disease Burden and Epidemiology

Epidemiology

The emergence of the pandemic HINI influenza virus in humans in early April 2009 in Mexico and California came as a totalsurprise. The virus first emerged in a little village in Vera Cruz, Mexico, but went unnoticed as no case of illness requiredhospitalization. The first two cases in California occurred in a 10 year-old boy and a 9 year-old girl who both necessitatedhospitalization. The H1N1 strain then quickly spread worldwide through human-to-human transmission. The number of countries,overseas territories or communities that reported laboratory-confirmed A/H1N1 cases in humans was more than 207 onNovember 22nd, 2009. Most countries in the southern hemisphere reported more pandemic H1N1 in 2009 than any of the seasonalsubtypes. In the temperate areas of the northern hemisphere, the spread of the pandemic was more gradual, initially spreadingwidely in the USA, Spain, Great Britain, Japan and Germany before invading other countries as well in the Fall. In the tropics,rates appeared to be quickly increasing in countries in both Central and South America and Asia, especially in Thailand, but veryfew data exist regarding the African continent. It is not possible at this time to guess what will the future look like. The mostpessimistic estimates call for 1 billion to 3 billion people (15% to 45% of the world's population) getting infected.

On the basis of recorded clusters in the USA, the household secondary attack rate was estimated to be 27.3%. In schooloutbreaks, a typical schoolchild infected on average 2.4 (range 1.8-3.2) other children within the school. The basic reproductivenumber, R0, thus ranged from 1.3 to 1.7 [423] . This is consistent with further pandemic spread causing illness in 25% to 39% ofthe world's population over a 1-year period, similar to the spread of the 1957-1958 Asian influenza A (H3N2) pandemic.

The actual number of influenza A (H1N1) cases worldwide remains unknown, as most cases are diagnosed clinically and are not


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laboratory-confirmed [424] but it most probably is in the order of several millions cases. In most countries, the capacity forlaboratory diagnosis has been so severely stressed that virological surveillance had to be restricted to patients attending hospitals[425] . The number of influenza-like illnesses during the 2009 spring outbreak in New York City has been estimated at 750 000-1 million cases.

Age distribution

A characteristic feature of the H1N1 pandemic is that it disproportionately affected so far children and young adults [426] . Oneof the early American studies showed that, although the age of H1N1 patients in the study ranged from 3 months to 81 years,60% of patients were 18 years of age or younger [427] . In most countries, the majority of H1N1 cases have been occurring inyoung people, with the median age estimated to be 12 to 17 years in Canada, the USA, Chile, Japan and the UK. Of the 272patients with 2009 H1N1 influenza who were hospitalized in the USA from April to mid-June 2009, 45% were under the age of18 years, whereas 5% only were 65 years of age or older [428] . This age distribution speaks in favor of at least partial immunityto the virus in the older population.

Of note, however, is the fact that if the highest rate of severe disease leading to hospitalization has been in the less than 5 yearsof age, the highest case fatality rate was recorded in the 50-60 years-old population. Subsequent studies have shown that 33% ofhumans over 60 years of age had cross-reacting antibodies to S-OIV A(H1N1) by hemagglutination inhibition test andneutralization tests, but antibody titers to A/H1N1 did not substantially increase after vaccination with a seasonal vaccine, evenwhen formulated with water-in-oil adjuvants [417] [429] . In another study, no neutralizing antibodies against the pandemicA/H1N1 (2009) virus could be found in sera from people born after 1920 [430] . However, a strong conservation of more than50% of T cell epitopes (whether T-helper or CTL epitopes) was described between the 2009 A(H1N1) S-OIV and the seasonalinfluenza virus strains used to prepare the 2007 and 2008 influenza vaccines, which would provide a definite level of cross-reactive cellular immunity to the pandemic virus in the vaccinated human population [431] . In addition, the possible role of theNA antigen in cross-protective immunity, which remains poorly explored, should not be forgotten [432].

Clinical presentation and severity of the disease

H1N1 is most of the times a rather mild, self-limiting upper respiratory tract illness with (or at times without) fever, cough andsore throat, body aches, fatigue, chills, rhinorrhea, conjunctivitis, headache and shortness of breath. Up to 50% of patientspresent with gastrointestinal symptoms including diarrhea and vomiting. The spectrum of clinical presentation varies fromasymptomatic cases to primary viral pneumonia resulting in respiratory failure, acute respiratory distress, multi-organ failure anddeath [433] . The H1N1 virus can bind alpha2-3 sialic acid receptors found on the surface of cells located deep in the lungs thatseasonal influenza virus cannot bind, suggesting why people with the pandemic flu can experience more severe pulmonarysymptoms [434].

Thus, 2% to 5% of confirmed cases in the USA and Canada and 6% of cases in Mexico had to be hospitalized, a fifth of themrequiring management in intensive care unit (ICU). The rate of hospitalization could actually be as high as 10% in some cities.Most, but not all of the hospitalized patients had underlying conditions such as cardiovascular disease, respiratory diseaseincluding asthma, auto-immune disorders, obesity, diabetes or cancer [428].

Pregnant women, especially in their second and third trimester, also are at a high risk for severe disease [422] [435] . Thus,more than one-third of the pregnant women with confirmed H1N1 infection had to be hospitalized during the current pandemic inthe USA due to acute respiratory distress syndrome [436] . Similar findings were reported from Australia and New Zealand,where the number of ICU admissions due to influenza A in 2009 was 15 times the number due to viral pneumonia in recentyears: infants from 0 to 1 year of age and adults 25 to 64 years of age were at particular risk, as well as pregnant women, adultswith a body mass index greater than 35, and indigenous Australian and New Zealand populations. In-hospital mortality was 16%[437].

The overall H1N1 case fatality rate in Mexico was estimated to be around 0.4% [438] . The average case fatality rate that canbe deduced from the laboratory-confirmed cases officially reported to WHO as of 06 August 2009 was much lower (0.08%).Current estimates put the average case fatality rate at 0.15% to 0.25%. H1N1 deaths mostly occur in middle-aged adults (medianage around 40-50 years), contrary to seasonal Influenza where fatal disease occurs most often in the elderly (>65 years old).Most of the deaths occurred in patients with an underlying medical condition, but close to one third of the hospitalized patientswho died had no known underlying medical condition. In South Africa, a majority of fatal cases occurred in HIV-infectedpeople, including pregnant women. In a recent study of the first 16 weeks of the pandemic in California, which saw 1088 casesof hospitalization or death, the median age of hospitalized patients was 27 years of age, but the case fatality rate (11%) was


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definitely the highest in persons 50 years of age and older [439].

From data on medically attended and hospitalized H1N1 patients in Milwaukee and informations from New-York City hospitalson numbers of hospitalizations, use of intensive care units (ICU) and deaths, it was estimated that about 1 in 2000 (between 1 in4000 and 1 in 1000) people in the USA who presented with symptoms of pandemic H1N1 influenza infection died; about 1 in 400symptomatic cases required treatment in ICU; and 1 in 70 required hospital admission [440] . Among the medically attendedcases in Milwaukee, 60% were in the 5-17 years age group, but severity of the cases was by far higher in the 18-50 years agegroup. Quite higher figures have been reported elsewhere for the H1N1 case fatality rate [441] [442] , most likely reflecting alarge incertitude on the true numbers.

The official number of deaths from H1N1 infection worldwide that had been reported to WHO on November 22, 2009 was 7820.

During the pandemic outbreak in Mexico, an estimated 150 000 cases of influenza-like illness with 3312 hospitalizations occurredin metropolitan Mexico City. The economic impact of the pandemic was estimated as >$3.2 billion (0.3% of gross nationalproduct) [443].

Transmission

The modes of transmission of the pandemic A (H1N1) 2009 virus appear to be similar to those of seasonal influenza viruses andinvolve primarily close unprotected contact with large respiratory droplets. The contribution of close range exposure to small-particle droplets expelled when an infected person coughs is unknown but could be more prominent under special conditions suchas aerosol-generating procedures. The virus is also likely transmitted through contacts with fomites that are contaminated withrespiratory or possibly gastrointestinal fluids [444] . Many S-OIV-infected patients experienced diarrhea, and viral RNA canreadily be detected in the feces of these patients, making the potential for fecal-oral transmission a risk to take seriously intoaccount [427].

The incubation period for S-OIV infection appears to range from 2 to 7 days, but most patients probably shed virus from day 1before the onset of symptoms through 5 to 7 days after. Studies of transmission in animal models show that the H1N1 virustransmits just as efficiently as seasonal flu [445] , contrary to earlier findings at the start of the pandemic [446].

Virology

The novel S-OIV A/H1N1 2009 can be grown in canine kidney (MDCK) cell cultures or primary human airway epithelial cellcultures, or in embryonated chicken eggs. Scanning electron microscopy revealed virions of remarkably filamentous shape [430].

Sequence analyses showed the absence of markers associated with high pathogenicity in avian or mammalian species, such as amultibasic hemagglutinin cleavage site [447] or a lysine residue at position 627 in the PB2 protein [448] . The occurrence of amutation at position 222 in the HA gene segment of H1N1 isolates from post mortem specimens has been recently reported invarious countries including Norway, the USA, China, Japan, Brazil and France. Although suspected to be associated withincreased pathogenicity, this mutation did not change the antigenicity of the virus nor its susceptibility to anti-viral drugs, nor didit appear to provide the virus with increased transmissibility.

Molecular and antigenic characterization

Phylogenetic analyses of A (H1N1) virus isolates reveal a great homogeneity of genomic sequences. The virus is antigenicallydistant from human seasonal influenza viruses but genetically related to three viruses that circulate in pigs [418] [449] , with theHA (H1), NP and NS gene segments coming from the classical swine H1N1 lineage. The H1 sequence could actually be tracedback to the 1918 H1N1 pandemic virus (the "Spanish flu"), which has remained endemic in swine and continued to circulateamong pigs in Asia, the America's and until the 1980s also in Europe [422] [450] [451].

The NA (N1) and M genes of the 2009 H1N1 pandemic virus come from the 'avian-like' Eurasian swine H1N1 lineage, whichemerged in Europe in 1979 after reassortment between a classical swine and an avian H1N1 virus, then spread through Europeand Asia [452] , displacing the classical swine H1N1 virus from Europe and generating new reassortants in swine with differenthuman origin influenza A viruses [453].

Finally, the PA, PB1 and PB2 genes of the 2009 pandemic H1N1 virus are from the North American H3N2 'triple-reassortant'lineage, which was first isolated from American pigs in 1998 in which it showed unusual pathogenicity [454] [455] [456] . The


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name 'triple-reassortant' relates to the fact that the virus has genes of human influenza virus origin, of classical swine influenzavirus origin and of North American avian virus origin.

The 2009 S-OIV H1N1 therefore has inherited virus gene segments of all three swine, human and avian origin: its HA, NP andNS gene segments have been inherited from swine classical virus, its NA and M segment from the avian-like Eurasianreassortant lineage, its PB1 segment from human H3N2 virus, and its PA and PB2 segments from North American avian lineage.Indeed, all gene segments of the pandemic A (H1N1) S-OIV were already established in the North-American 'triple-reassortant'(H3N2) swine virus and in the 'avian-like' Eurasian (H1N1) swine virus but no data are available to help evaluate when, where,nor between which parent viruses did the initial reassortment actually occur [417] [419] [420].

Antigenically, all S-OIV isolates look similar to classical swine viruses and to reassortant H1N1 viruses that have beencirculating among pigs in the USA over the last decade, showing no antigenic cross-reactivity with contemporary human seasonalH1N1 viruses. Surprisingly, there is no evidence that pigs play any role in the epidemiology or in the worldwide spread of thepandemic A (H1N1) virus in the human population [421].

Pathogenicity in animals

Experimental pathogenicity of the 2009 A/H1N1 S-OIV was tested in mice, ferrets and nonhuman primates [430] . S-OIVreplicated more efficiently in the lungs of infected mice, generating earlier bronchitis and alveolitis, and eliciting more markedlyincreased production of interleukin-10 (IL-10), interferon gamma (IFN-Y), IL-4 and IL-5 than infection with a recent humanH1N1 virus (A/Kawasaki/UTK-4). Similarly, it induced in nonhuman primates more elevated fever, more severe lung lesionswith oedematous exudate and inflammatory infiltrates and higher antigenic loads in pneumocytes, similar to what was reportedfor highly pathogenic avian H5N1 influenza viruses [457] . This may have to do with affinity of the virus for alpha2-3 sialic acidreceptors in the lower respiratory tract [434] . The pandemic virus also was more pathogenic in ferrets, replicating to highertiters in trachea and lungs than human seasonal H1N1 virus and caused more severe bronchopneumonia with prominent viralantigen expression in the peribronchial glands and alveolar cells. In contrast, it was devoid of overt pathogenicity forpathogen-free miniature pigs, although the virus did replicate efficiently in the respiratory tract of the animals.

The 2009 A(H1N1) S-OIV was also found to be more pathogenic than a seasonal 2007 A (H1N1) virus in ferrets and mice, withextensive virus replication occurring in the trachea, bronchi and bronchioles of the animals, while replication of the seasonalvirus was limited to the upper respiratory tract [458] . The 2009 A(H1N1) influenza virus also replicated in the intestinal tract ofinoculated ferrets, consistent with gastrointestinal involvement in many human A(H1N1) cases [446]. Transmission of the virusvia aerosol or respiratory droplets was also tested in ferrets, and found to be either as efficient as [458] or less efficient than[446] highly transmissible seasonal A(H1N1) virus. The latter observation is in agreement with the observation that the virus maynot be that easily transmissible among humans as only 10% of patients' household contacts become infected [459].

Sensitivity to antiviral drugs

Genetic and phenotypic analyses indicate that S-OIV is susceptible to neuraminidase inhibitors oseltamivir and zanamivir, butresistant to the adamantanes [460] . Treatment with oseltamivir is efficacious if initiated within the first 36 hrs after infection.The FDA issued an emergency use authorization approving the use of oseltamivir to treat influenza illness in infants under theage of 1 year and for chemoprophylaxis in infants older than 3 months of age.

Close to one hundred A(H1N1) S-OIV isolates have been described that were resistant to oseltamivir. These cases have beensporadic and there was no evidence of further transmission of the resistance marker into the virus population.

Vaccines

Vaccines are considered to be one of the most effective tools, not only to prevent the spread of the influenza virus but also tomitigate the severity of illness and the impact of the disease [461] . Today, the implementation of a pandemic A(H1N1)influenza vaccine in the fastest time is a global priority. This stems both from the rapid spread of the pandemic worldwide, fromthe fear that the A(H1N1) virus might accidentally gain added virulence through mutations and/or reassortment with other humanor avian influenza virus, and from the total lack of cross-immune reactivity observed between the pandemic and seasonalinfluenza virus strains, making the 2009 seasonal vaccine useless in the fight against the A(H1N1) pandemic.

The development of a pandemic influenza vaccine however raises complex challenges, such as ensuring that sufficient seasonalinfluenza vaccine will still be available in due time, estimating with accuracy short- and medium-term production capacity of the


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different producers, reserving part of the foreseen production capacity for under-resourced countries with no or little access tothe vaccine, etc [462] [463].

As of June 2009, the total global annual capacity for trivalent seasonal influenza vaccine production stood at 876 million doses,with seven manufacturers responsible for 560 million doses (i.e. 64% of the capacity). In spite of the WHO global pandemicinfluenza action plan to increase the potential supply of pandemic influenza vaccine [464] , the supply of enough pandemicvaccine to immunize the world's population -should this be needed- would therefore take at least four years! Added to which, itwas not clear at that early time whether one or two doses of pandemic vaccine would be required to induce full protection, norwhether the use of water-in-oil adjuvants would have the same antigen dose-sparing effect as in the case of the H5N1 vaccines[465] [466] . Finally, the yields of virus production in eggs or cell cultures, which is an important determinant of the amount ofvaccine doses that can be manufactured, were not quite up to expectation.

A total of 26 vaccine manufacturers from America, Europe, Russia, Australia and Asia have now developed or are presentlydeveloping pandemic A(H1N1) vaccines, whether inactivated whole-virus vaccines, split inactivated vaccines, subunit vaccines,live-attenuated vaccines or other formulations. Of note is the participation of new vaccine manufacturers in China, India,Thailand and South America. All H1N1 vaccines were tested in clinical trials for safety and immunogenicity. Clinical trials stillare in progress in certain at-risk subpopulations.

Preliminary reports indicated that a single 15-µg dose of an inactivated split influenza A (H1N1) 2009 vaccine induced ahemagglutination-inhibition assay titer of 1:40 or more in 96.7% of 18-64 years-old subjects [467] . The robust immune responseobserved in the 18-49 years-old volunteers cohort was unanticipated, suggesting that there is more similarity between theinfluenza A (H1N1) 2009 virus and recent seasonal virus strains than had been recognized so far [468] . The NIAID Office ofCommunications also reported that among healthy volunteers who received a single 15-µg dose of either the Sanofi-Pasteur or theCSL Limited inactivated split A (H1N1) 2009 vaccine, a robust immune response was measured in 96% and 80%, respectively,of adults aged 18 to 64, and in 56% and 60%, respectively, of adults aged 65 and older [469].

In a recent Phase II trial on 410 children and 724 adults who received a single - dose (15 µg HA) of inactivated A(H1N1)vaccine in the USA, protective serological titers of >1:40 were detected at 21 days after vaccination in 45%-50% of 6-35month-old infants, 69%-75% of 3-9 year-old children, 95-100% of 18-64 year-old adults, and 93%-95% of elderly adults [470] .No vaccine-related severe adverse event occurred, but about 50% of every age and vaccine group reported injection-site andsystemic reactions. Similarly, a multi-center, double-blind, randomized trial on 12691 3 years of age or older persons receiving asingle-dose (7.5 µg HA) of a split virion A/H1N1 vaccine in China showed that protective serological titers were detected on day21 in 76.7% of 3-12 year-old children, 96.8% of 12-18 year-old adolescents, 89.5% of 18-60 year-old adults, and 80.3% of adultsolder than 60 years. In children, the administration of a second dose of the 7.5 µg formulation increased the seroprotection rate to97.7% [471].

The fact that it is possible to induce in adults protective antibody levels against A (H1N1) infection within two weeks ofadministration of a single dose of vaccine has now been confirmed with every pandemic H1N1 vaccine tested [472] , whethersplit inactivated vaccines containing 15 µg HA (SanofiPasteur, CSL, Sinovac and others), split inactivated vaccines with awater-in-oil adjuvant containing either 7.5 µg HA and MF59 (Novartis) [473] or 3.8 µg HA and AS03 (GSK), or whole-virionvaccines containing 10 µg HA (Baxter) or 6 µg HA (Omnivest, Hungary) [474] . The same one-dose schedule applies tointranasal live attenuated influenza virus (LAIV) vaccines (MedImmune in the USA and Microgen in Russia). Nationalauthorities have recommended that young children should receive a two-dose schedule, as is the case for seasonal vaccines, butimmunogenicity data from clinical trials indicate that with many vaccines a single dose induces appropriate levels of immuneresponses.

Vaccination against pandemic H1N1 influenza was first implemented in China [475] , followed by a large number of countries.The problem still remains, however, of vaccinating the populations living in under-resourced countries, which cannot afford tobuy the vaccine, and which depend on donations from governments of industrialized countries and from the pharmaceuticalindustry. The WHO is efficiently coordinating this effort.

[476] are health care workers and pregnant women, whose vaccination is a highly cost-effective strategy with substantialbenefits to both the infants and the expectant mothers [477] [478] . Other priority groups are individuals with an underlyingcardiovascular or respiratory medical condition including asthma, auto-immune disorders and diabetes, as well as young children.

The safety of the pandemic H1N1 vaccines has been thoroughly monitored during the various clinical trials. Current data showthat the vaccines are well tolerated and behave as the corresponding seasonal vaccines in terms of safety and lack of severe


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adverse events. A small number of Guillain Barré syndromes have been reported after H1N1 vaccine administration inlarge-scale vaccination campaigns, but they all reverted quickly. Although oil-in-water adjuvanted vaccines have been approvedfor use in all populations by the European Association EMEA, including pregnant women, their use in the USA raises regulatoryproblems, as no adjuvanted flu vaccine has ever been licensed in the country and as no fast-track system is in place for theirregistration [479].

The emergence of the 2009 H1N1 pandemic and its global impact on Public Health have revived the dream of a 'universal'influenza vaccine that could provide solid, broad subtypic protection against influenza viruses and skip the need for yearlyseasonal vaccinations. The recent finding that the human immune system can recognize a conserved neutralization epitope on theHA molecule that is shared across several influenza virus subtypes [480] [481] , combined with the fact that the well-conservedNP viral nucleoprotein could generate cross-protective cellular immunity [482] are strong arguments in favor of the possibility ofdeveloping such a vaccine [483] . It also has been shown that the conserved external region of the ion channel M2 viral protein(M2e) can elicit cross-protection through antibody-dependent cellular cytotoxicity (ADCC) [484] [485] [486] [487] . However,pigs which were vaccinated with a human influenza M2e vaccine [487] showed more severe clinical signs than non-vaccinatedcontrol animals when challenged with a swine H1N1 influenza virus. Three out of six vaccinated pigs died after challenge,suggesting that antibodies to human M2e, especially in combination with cell-mediated immune responses, exacerbated swineinfluenza disease [488] . These results cast doubt on the feasibility of using safely M2e as an immunogen in humans.

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