The challenge of enteric fever

Journal of Infection (2014) 68, S38eS50

www.elsevierhealth.com/journals/jinf

The challenge of enteric fever Claire S. Waddington, Thomas C. Darton, Andrew J. Pollard* Department of Paediatrics, University of Oxford, Children’s Hospital, Oxford OX3 9DU, United Kingdom Accepted 20 September 2013 Available online 8 October 2013

KEYWORDS Enteric fever; Typhoid; Paratyphoid; S. Typhi; S. Paratyphi; Controlled human infection; Challenge studies; Disease control; Vaccine

Summary Enteric fever, a non-specific, systemic infection caused by S. Typhi or Paratyphi A, B or C, is common in resource-limited regions of the world, where poor sanitation infrastructure facilitates faeco-oral transmission. Prompt treatment with appropriate antibiotics minimises illness severity, but presentation to health care facilities is often delayed because of the non-specific nature of the symptoms and the lack of reliable diagnostic tests. Disease prevention requires significant investment in provision of clean water and sanitation in the long term; vaccination offers a more realistic strategy for medium term control. However, implementation of existing vaccines and development of more efficacious vaccines has been hindered by the lack of an established correlate of protection and under appreciation of the true disease burden. Human microbial infection studies could provide a vehicle for the rapid evaluation of novel vaccines and investigation of the immunobiology of enteric infection. ª 2013 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

Introduction Enteric fever is a systemic infection caused by Salmonella enterica subspecies enterica serovars Typhi (S. Typhi, causing typhoid fever) or Paratyphi A, B or C (S. Paratyphi A, B or C, causing paratyphoid fever). Enteric fever is common in resource-poor regions of the world, where poor sanitation and inadequate clean water provision facilitate the spread of infection via faeco-oral transmission. Enteric fever produces a wide range of non-specific symptoms and is clinically indistinguishable from many other diseases, both infectious and non-infectious. Prompt

treatment minimises illness severity, but late presentation to health care facilities often delays initiation of appropriate antibiotic therapy. Treatment delay is compounded by a lack of reliable diagnostic tests. Although typhoid could be controlled by improvements in public infrastructure (for example, clean water and separate sewage systems), the significant funding investments required in many regions are unlikely to be forthcoming in the near future. Fortunately, disease control in the interim may be possible through effective vaccination. Indeed, prevention of typhoid fever has been attempted through vaccination for over 100 years.1 Three licensed

* Corresponding author. University of Oxford, Room 02-46-07, Level 2, Children’s Hospital, Oxford OX3 9DU, United Kingdom. Tel.: þ44 (0) 1865 231693. E-mail addresses: [email protected] (C.S. Waddington), [email protected] (T.C. Darton), [email protected] (A.J. Pollard). 0163-4453/$36 ª 2013 The British Infection Association. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jinf.2013.09.013

Enteric fever vaccines for typhoid fever exist, but are only moderately efficacious and unsuitable for infant immunisation. There are currently no licensed vaccines to prevent paratyphoid infection. The implementation of existing vaccines and development of new efficacious vaccines has been hindered by a lack of understanding of typhoid immunobiology. S. Typhi and Paratyphi are human restricted pathogens and require a relatively high dose to cause infection. Consequently the development of a suitable vaccine could lead to the eradication of enteric fever. To reach this goal, an improved understanding of the immunobiology of typhoid fever is needed.

Epidemiology Typhoid fever is estimated to affect at least 26.9 million people per year, of whom 1% will die.2 Paratyphoid fever has been estimated to affect 5.4 million people per year,3 and appears to be increasingly prevalent, with recent reports attributing up to half of all enteric fever cases to paratyphoid infection in some Asian countries.4,5 The incidence of paratyphoid fever in returning travellers is also increasing.6,7 The majority of the global disease burden is borne by children and adolescents in resource-poor settings, particularly in Asia.8 The distribution of disease is changing however, with increasing recognition of a significant disease burden in Africa.2,9 The true burden of enteric fever may be considerably higher than these estimates suggest.2,8,10 There are significant limitations to the data used to generate morbidity and mortality estimates. Resource-poor countries frequently lack the health care and public health infrastructure to provide reliable data.3,8,11,12 Historically, cases of clinical enteric fever have been attributed to S. Typhi but with the increasing burden of paratyphoid fever in many regions, this is unreliable.13 Data from population-based blood culture studies, although specific, are few in number.14 These studies consistently demonstrate a higher disease prevalence than that suggested by public health figures.12 Similarly, sero-epidemiological studies based on detection of antibodies to typhoid antigens suggest higher rates of infection than studies using clinical or microbiological case detection, with up to 80% of residents in endemic regions showing evidence of past infection.15,16 Microbiological isolation of S. Typhi or S. Paratyphi provides a definitive diagnosis, but blood culture sensitivity is limited to 50%e 60% and therefore underestimates the true number of cases,17 and bone marrow culture is rarely available. Traditional assessments of enteric fever disease burden have focused on Asia; the incidence of disease in Africa and South America remains uncertain, although recent reports suggest it may be an increasingly recognised problem. Asian countries have previously been estimated to account for 93% of the known worldwide mortality and morbidity,8 with current estimates of 394.2 cases per 100,000 population.2 Correspondingly, the majority of infected travellers are returning from Asia. For example, in laboratory-based surveillance of US isolates, 65% of patients had travelled to one of four countries e India, Pakistan, Bangladesh or Haiti.18 Encouragingly, the disease burden in some parts of Asia may be falling, particularly in areas where significant

S39 investments in infrastructure have been made. A 15 year retrospective review of blood culture results in Vietnam showed that, while S. Typhi had accounted for 74% of positive blood cultures in 1994, the proportion by 2008 was 6.2%.19 The marked decline in malaria prevalence in many regions of Africa over the last decade has promoted S. Typhi to one of the leading causes of severe febrile illness. A meta-analysis of community-acquired bloodstream infections across Africa estimated that S. Typhi accounted for 9.9% of all isolates.9 Although this meta-analysis extrapolated limited data across a diverse continent and people, it has been supported by other data. In children under 15 years of age in Ghana, 12.4% of blood culture isolates were due to S. Typhi, equivalent to an incidence of approximately 190/100,000 per year.20 Similarly, in Nigeria, S. Typhi accounted for 20.9% of isolates in children less than 5 years of age, despite high pre-culture antimicrobial use.21 Disease rates in Zanzibar match those seen in many high incidence regions of Asia, with 58% of blood culture isolates identified as S. Typhi.22 Although these data confirm the importance of typhoid in Africa at the clinical front-line, the false attribution of febrile illness to malaria has often hindered the recognition of enteric fever as a major cause of disease in Africa.9,21 Comparison of blood culture isolates with clinical diagnoses in Africa showed that 55% of children with S. Typhi bacteraemia were diagnosed clinically with malaria,21 supporting the idea that enteric fever is an under recognised problem in Africa and highlighting the need for better diagnostics and preventative measures in resource-poor countries.

Acute typhoid fever Enteric fever is a highly variable, non-specific illness. Typhoid fever and paratyphoid fever cannot be distinguished on clinical grounds.6 Fever is the most frequent and universal symptom with other frequent symptoms being malaise, chills, anorexia, diarrhoea, headache, weight loss, abdominal pain and rash.23,24 Nausea, constipation, myalgia, arthralgia and cough are also reported.25e27 Severity of symptoms is highly variable, with some patients able to continue normal activity and some requiring inpatient care.27 Specific physical signs are frequently absent, but diffuse abdominal tenderness is common, and hepatosplenomegaly and/or lymphadenopathy may also be present.12,24 Rose spots, a transient, fine, blanching maculopapular rash, usually starting on the trunk and spreading to the arms and legs, are pathognomonic for typhoid fever, but is reported with a range of frequency from as few as 3% of cases, up to 40%.12,24 A relative bradycardia is also described.12 S. Typhi and Paratyphi infection can lead to a variety of other rarer clinical entities including meningitis, septic arthritis and osteomyelitis.28

Chronic carriage In 1902, Robert Koch postulated that healthy people could carry disease causing organisms and serve as reservoirs of infection.29 Applying this idea to typhoid fever, Koch noted that humans were the only source of S. Typhi, and

S40 hypothesised that a carrier state could bridge the gap between one typhoid outbreak and the next. Prospective studies of patients with typhoid fever in Germany confirmed this, with identification of ongoing excretion of S. Typhi in the stool of symptomatic individuals.29 The occurrence of typhoid carriage in people without a history of illness was documented shortly after, with chronic carriers described by one author as “living storehouses and factories of disease”.30 The role of chronic carriers in disease transmission was famously illustrated by the case of the household cook Mary Mallon (Typhoid Mary) in the early part of the 20th century.29 Mary Mallon infected between 26 and 54 people, before being requested by public health officials to cease working as a cook.31 Mary Mallon continued to seek employment as a cook however, and was eventually incarcerated until her death, on the grounds of the risk to public health. Sadly, Mary Mallon was not alone in this and incarceration of chronic carriers continued throughout the 20th century. In 2008 in the United Kingdom it emerged that 43 female typhoid carriers had been housed for life in an asylum at various points between the asylum’s opening in1907 until its closure in 1992.32 Although famous, the extent of disease spread from Mary Mallon was modest compared to similar outbreaks resulting from a single carrier. For example, in the UK a large outbreak spanning 13 years was eventually traced in 1909 to a cowman and milker referred to as Mr N29 who had worked at five farms that directly supplied milk, resulting in 207 cases of typhoid fever, 64% of the total cases reported in that region during that period. Surveillance of patients with acute typhoid fever has shown that up to 10% of untreated patients excrete S. Typhi in their stools for three months after infection.12 Between one and four per cent of patients continue to excrete S. Typhi beyond this, and are considered to be chronic carriers.13 Carriers of S. Typhi have been shown to excrete between 106 and 1010 organisms per gram of faeces,33 a large bacterial load that facilitates the faecal-oral route of transmission. This is especially true of chronic carriers who work as food handlers, as illustrated in the historical cases above. Indeed, during Mr N’s employment as a sailor there was no evidence of secondary spread.29 Gallstones are thought to be the principal risk factor for developing chronic carriage,34 and are detected in approximately 90% of chronic carriers.35 Gallstones allow attachment of the bacteria and biofilm formation,36 which in turn protects the bacteria from the antimicrobial and emulsifying action of bile.37 The need to treat chronic carriage, for both public health protection and the prevention of biliary cancer, seen as a sequel to chronic carriage,38 is clear. Treatment strategies remain limited however. A trial of high dose ampicillin for 28 days was shown to cure five out of six chronic carriers, and provided hope that the condition could be effectively treated with prolonged courses of antibiotics.39A further trial of 12 patients showed a 75% cure rate 90 days of ampicillin, but 66.7% experienced reactions to the high doses of antibiotics required.40 Successful eradication of chronic infection with ciprofloxacin has also been reported,41 but antibiotic therapy alone does not lead to cure in all patients, probably due to biofilm formation by

C.S. Waddington et al. S. Typhi on gallstones preventing antibiotic penetration.42 In patients with gallstones cholecystectomy is an effective long term solution,42 with a cure rate of approximately 75%. Combining surgery and antibiotics improves the outcome further to greater than 90%,43 but carriage still persists post-operatively in some patients.44,45 Surgical intervention is a relatively expensive option and remains unavailable to many patients in typhoid endemic regions. Prevention of disease transmission and thus the initial infection will require either improved provision of sanitary water or effective general vaccination or both to be achieved.

Diagnosis Prompt diagnosis and treatment of typhoid fever decreases disease complications and limits opportunity for disease spread.12 Achieving a prompt and reliable diagnosis is difficult however. Enteric fever leads to a diverse range of clinical symptoms and signs, and cannot be reliably distinguished on clinical grounds from other diseases that are common in endemic regions, including malaria, tuberculosis, dengue fever and brucellosis.12 Diagnostic tests that are both sensitive and specific are lacking, especially in endemic regions, where laboratory facilities are frequently limited.46 Novel, reliable diagnostic tests are needed to prevent delays in treatment, minimise morbidity and mortality, prevent inappropriate use of antimicrobials and provide reliable epidemiological data. The discovery by Widal in the late 19th century that sera from patients with typhoid fever agglutinated typhoid bacilli formed the basis of the Widal test, which has been used for the diagnosis of typhoid fever ever since.47 The current Widal test detects the titre of agglutinating antibodies to the O (lipopolysaccharide) and H (flagella) antigens by serial dilution.48,49 The demonstration of a fourfold rise in antibody titre between acute and convalescent samples is considered diagnostic.49 However, the Widal test and other serological tests are limited by the high residual background levels from previous positive results in endemic regions, and cross reactivity with other antigens49e55; in addition, paired acute and convalescent samples are frequently not available. Furthermore, serological tests do not distinguish S. Typhi from S. Paratyphi infection. Newer generation serological tests have been developed, but have failed to overcome the limitations of the Widal assay.56 Culture of S. Typhi or Paratyphi from blood or bone marrow is diagnostic, but is not achieved in all patients.17 Blood culture has been employed as a diagnostic method in typhoid fever for over 100 years57 and is clinically acceptable, provides a definitive diagnosis when positive and is relatively easy to perform where basic microbiology facilities are available. Culture sensitivity is limited however, in part due to the low bacterial load in blood during acute typhoid fever, with a median count of one colony-forming unit per ml of blood.58 Bacterial load decreases further with increasing duration of illness58,59 reducing the detection rate when presentation is delayed.59 Sensitivity is variable between 30% and 90%59e61 and several days of incubation may be needed before a positive result is obtained.

Enteric fever Culture of bone marrow is more sensitive than of blood, and is the gold standard diagnostic test.62 Sensitivity rates of 90% are reported, even with prior antimicrobial administration.60,61 Concentrations of bacteria in the bone marrow are higher than in blood, possibly accounting for the increased sensitivity.17 Bone marrow culture is limited by invasiveness and the requirement for technical skill and is therefore rarely used in clinical practice10,13 but may be useful if repeated blood cultures are negative, or if previous antibiotic therapy has been used. An alternative approach to the diagnosis of enteric fever is the use of PCR to detect microbial DNA. The low concentrations of microbial DNA in clinical samples has proved limiting however. The amount of mammalian DNA greatly exceeds the amount of bacterial DNA, potentially leading to false-positives resulting from non-specific amplification, as has been shown with assays based on the detection of 16s rDNA from microbes.63 The volume of blood available from patients is often small, especially among children. To overcome these problems, a novel approach was used by Zhou et al.64,65 A short period of culture in ox-bile, previously shown to be the optimal culture media for S. Typhi,66 was combined with a PCR assay, targeting the flagellin gene.64 At concentrations up to 2.4%, the bile-containing media facilitates sustained growth of Salmonella Typhi whilst lysing blood cells, should theoretically liberate the DNA of these intracellular organisms and inhibit the bactericidal action of blood. After a minimum of 3 h incubation this approach detected DNA in 4 ml of blood spiked with 3 CFU of S. Typhi showing that this combined approach is rapid but sensitive, and offering hope that this method can be used for reliable diagnosis in patient samples.

Preventing typhoid fever Sanitation and clean water Endemic enteric fever results from a lack of basic hygiene and sanitation infrastructure which facilitates disease spread. The importance of sanitation and clean water in the prevention of endemic typhoid fever has long been recognised.67 During the early part of the 20th century, resource-rich countries saw a marked decline in the incidence of typhoid fever following the routine provision of sanitation and clean water, along with the advent of antibiotics that limited infectivity.68,69 However, high disease rates are still seen in resource-poor countries where such infrastructure and intervention are still lacking.70 Seasonal variation in the incidence of endemic typhoid fever is reported, and has also been related to water sanitation. The nature of this variation is variable from region to region.10,71 In Pakistan, Indonesia and Vietnam, the incidence of disease has been found to peak during the dry season when ambient temperatures rise after the rainy season, and the quality of water deteriorates due to a lack of fresh supplies.71e73 The increased incidence in hotter months may also reflect an increase in the consumption of ice cream and iced drinks, a risk factor for typhoid fever.74,75 In other regions, typhoid incidence is reported to increase in the rainy season.76,77 This may be due to

S41 high water flow overwhelming sanitation systems that separate sewage from clean water.77 The typhoid fever epidemic in Tajikistan (1996e7) further highlighted the influence of water contamination as the principle mode of transmission. During a period of civil unrest, standards of water chlorination decreased.78 In a six month period, 1% of the capital city’s population were affected by typhoid fever78 and the epidemic abated when measures to improve water standards were implemented. This outbreak illustrates the dramatic effect that the loss of clean water supplies can have and the extent to which epidemics of typhoid fever can affect a population.

Travel-associated enteric fever In resource-rich countries, the percentage of all cases of typhoid fever which are attributable to travel is rising decade by decade,18,79 although the rates of cases per thousand travellers has decreased.80 In laboratory-based surveillance in the United States, 81% of isolates were recovered from patients with a travel history, especially those who had visited Asia.18 A separate review of cases reported in the period from 1994 to 1999 in the United States attributed 74% to travel, of which 53% were acquired in the Indian subcontinent.81 In the UK there has been a steady increase in the number of enteric fever cases since 1996, with a notable increase in the proportion attributed to S. Paratyphi A.7 Returning individuals who have travelled to endemic countries to visit friends and relatives (VFRs) account for around 85% of cases in the UK;82 these individuals are also the least likely to have taken pre-travel advice and related precautions. Vaccination against typhoid fever is recommended for most travellers over 18 months of age who are travelling from the UK to countries in Africa, South Asia, the Middle East, Central and South America and the Caribbean.83,84 Efficacy studies for typhoid vaccines in the prevention of travel associated infection have not been conducted, but only a very small number of patients with travel related typhoid fever have received a vaccine prior to travel79,81 suggesting that failure to vaccinate rather than failure of the vaccines is more commonly the problem. For example, of the 294 cases of enteric fever reported in the UK in 2006e2007, only 54 had received a typhoid vaccine in the last 3 years. Among these only 12 cases of S. Typhi infection occurred, the remaining 42 had S. Paratyphi.82 There is a relatively high incidence in travellers aged less than two years of age (7% of cases in one series),81 emphasising the need to develop a vaccine suitable for this vulnerable age group. These findings highlight the role for established typhoid vaccines in the prevention of travel-associated enteric fever, and the need to educate travellers, especially those visiting friends and relatives abroad. The high incidence among travellers also demonstrates the urgent need for vaccines to prevent paratyphoid infection and for enteric fever vaccines that are suitable for infants and young children.

Current vaccination strategies Currently available vaccines and novel vaccines against typhoid and paratyphoid fever are shown in Table 1. Three

S42 Table 1

C.S. Waddington et al. Currently licenced vaccines and novel vaccines against typhoid and paratyphoid fevers. Vaccine type

Licensed vaccines against typhoid fever

Comments

Killed whole cell

Live oral attenuated

Vi capsular polysaccharide Novel vaccines against typhoid disease

Vaccine

Live oral attenuated

Vi polysaccharide eprotein conjugate

Lipopolysaccharide-conjugate vaccine (Ty21a)

Licenced vaccines against paratyphoid fever Novel paratyphoid Lipopolysaccharide e tetanus vaccines toxoid conjugate Live oral attenuated

Not currently manufactured. Rendered obsolete because of high rates of reactogenicity. Ty21a

Only manufactured in a formulation suitable for children6 years of age. Revaccination recommended every 5e7 years. Given as 4 doses (North America) or 3 doses (rest of the world) on alternate days. Licenced in children 18 months. Repeat immunisation every 3 years. M01ZH09 2 independently attenuating gene deletions in ssaV and AroC genes. Phase I and II studies completed.126,165 CVD 909 Mutations in aroC, aroD and htrA. Modified to constitutively express Vi antigen. Phase II studies completed.123 Ty800 Mutation in phoP/phoQ. Safe and immunogenic in phase I studies, but not further developed for commercial reasons.140 Vi-rEPA Safety and efficacy of 91.5% in children 2e5 years over 27 months demonstrated in phase III study.143e145 Vi-tetanus toxoid Licensed in India, but not prequalified by WHO. Vi-diptheria toxoid Currently in phase I trials. Vi-CRM197 Vi antigen derived from Citrobacter, minimising risks of manufacture. Phase II trials.146 LPS conjugated to Pre-clinical trials currently being conducted.152 diphtheria toxin. Cross protection of this typhoid vaccine has been demonstrated against S. Paratyphi B.153 Safe and immunogenic in children and adults.154 CVD 1902 phoPQ mutants

Bivalent typhoidparatyphoid vaccine

Vi polysaccharide of S. Typhi and polysaccharide of S. Paratyphi independently conjugated to carrier proteins.

distinct types of typhoid vaccine have been licensed: killed whole cell parenteral vaccines, a live attenuated oral vaccine (Ty21a) and Vi polysaccharide parenteral vaccines. All have their limitations and new vaccines are needed to overcome these. There are no licensed paratyphoid vaccines. Killed whole cell vaccines Vaccines designed to confer protection against typhoid fever were developed as early as the end of the 19th century.1,85 Early vaccines were killed whole cell vaccines, inactivated by heat and phenol or acetone. Heat inactivated typhoid vaccines were studied in several army units, but the studies were inadequate and evidence of efficacy was lacking.86 Despite this, heat inactivated typhoid vaccines were routinely used by the British army for many years.85

Live oral attenuated vaccine. Immunogenic in animals. In early human trials.156 Animal trials157 Preclinical trials with different conjugate proteins currently being undertaken.155

The true efficacy of killed-whole cell vaccines was first prospectively investigated in a series of WHO-sponsored randomised controlled trials in endemic regions in the 1950s and 1960s.87e96 The first of these trials was conducted in 1953 by the Yugoslav Typhoid Commission, and compared the heat-inactivated, phenol-preserved vaccine to the alcohol-inactivated and preserved vaccine.91 A control group received a vaccine against Shigella flexneri. The heat-phenol inactivated vaccine was demonstrated to be the more efficacious, with 70% protection against culture-confirmed typhoid fever.91 However, these vaccines were limited by their reactogenicity. Killed whole cell vaccines contain components of Gram negative bacteria, including lipopolysaccharide, that elicit profound systemic and local reactions due to the innate immune and inflammatory responses.97 Fever rates

Enteric fever after vaccination were approximately 20e25% and local reactions occurred in 40e50% of vaccinees.88,92,94 Reactions were of sufficient severity to lead to an absentee rate of approximately 15%.88,92,94 Despite this, these vaccines were successfully used to control typhoid disease in Thailand98 although they have now become obsolete.85,97,99 With the description in the 1940s of chloramphenicol as an effective treatment for typhoid fever, the pressure to develop suitable vaccination strategies was much reduced. The outbreak of chloramphenicol resistant strains in the 1970 once more focused attention on developing vaccines that could be introduced as part of public health programmes.100 Oral vaccine Ty21a A live, attenuated strain of S. Typhi suitable for use as an oral vaccine was first described in 1975.101 Referred to as Ty21a, the strain had been derived by nonspecific chemical mutagenesis of the parent wild-type S. Typhi strain Ty2, resulting in over 20 different mutations, of which the most significant were thought to be the disruption of the galE gene and the inability to produce the Vi capsular polysaccharide.101,102 Initial trials of the Ty21a vaccine were conducted in a human challenge model at the University of Maryland.103 After vaccination, volunteers were challenged with S. Typhi Quailes strain against which a protective efficacy of 87% was demonstrated.103 The first randomised, placebo- controlled, double-blind field trials of Ty21a were conducted in Alexandria, Egypt, between 1978 and 1981. These demonstrated that the Ty21a vaccine was both safe and had a protective efficacy of 95% in the field.104 Later trials in Chile showed lower efficacy of 67%105 but were able to demonstrate herd as well as individual protection.106 Through extensive trial experience with Ty21a and postlicensure data, the safety and tolerability of Ty21a is now well established.102 Several significant limitations exist however. The overall efficacy of the vaccine is moderate, with meta-analysis showing 51% protection.69 The current commercially-available Ty21a vaccine is a lyophilised formulation in enteric-coated capsules. This is difficult to administer to young children107 and is less immunogenic in infants when compared with older children.105 The vaccine has therefore not been licensed for use in children below 6 years of age. Although a “liquid” formulation of the vaccine (reconstitution of lyophilised vaccine buffer and water) has been shown to be practical, well tolerated and immunogenic,107 it is not currently being manufactured. Vi polysaccharide vaccine The virulence-associated (Vi) polysaccharide capsule of S. Typhi consists of a repeating homopolymer of alpha-1,4,2deoxy-2-N-acetylgalacturonic acid. It was first identified as a virulence factor by Felix in 1934.108 Killed whole cell vaccines containing the Vi antigen had been shown to be more efficacious than those that did not96 and therefore the potential of purified Vi as a vaccine was investigated. Following the successful isolation of non-denatured purified Vi polysaccharide99,109 (using the same detergent that had been used in the isolation of the polysaccharide for a meningococcal vaccine110), Vi was developed as a vaccine candidate.97 Subsequent safety studies showed that as

S43 long as contamination by LPS was minimal, the Vi vaccine was well tolerated.111 The vaccine was shown to generate anti-Vi antibodies in the majority of recipients and these antibodies persisted for at least three years.112 These initial safety studies were followed up with field trials to look at safety and efficacy. Having established safety in 274 people in Nepal as part of a pilot study, Acharya conducted a large trial involving 6438 participants between the ages of 5 and 44 years of age randomised to receive either a 25 mg dose of the Vi polysaccharide vaccine or pneumococcal polysaccharide vaccine as control.113 The vaccine was well tolerated, and around 75% of participants had a rise in anti-Vi antibodies of four fold or more.113 At the end of the study, vaccine efficacy was 72% against culture-positive typhoid fever.113 Similarly, a South African study comparing Vi capsular polysaccharide vaccine to meningococcal polysaccharide vaccine showed 60% protective efficacy against blood culture confirmed typhoid fever from day of vaccination, and 64% efficacy from 6 weeks post vaccination in a randomised- double blind trial of 11,384 school-aged children followed up with 21 months’ passive surveillance.114 The findings from these two field trials demonstrated that the Vi polysaccharide vaccine was safe and effective in endemic regions and the vaccine went on to be licensed in the United States in 1994.69 A recent cluster randomised trial of this vaccine also gave an indication of its potential to induce herd immunity; with a vaccine uptake of 60% in the Vi clusters, protective efficacy among unvaccinated members of the same Vi-clusters was 57%.115 However, significant limitations of the Vi polysaccharide vaccine remain. The administration of the vaccine via the parenteral rather than the oral route requires the use of trained health personnel and decreases the acceptability of the vaccine among target populations.116 As with other Tcell independent polysaccharide vaccines, the Vi polysaccharide does not generate immunological memory and is not boosted by repeated vaccination.117e119 The efficacy of the Vi vaccine in infants is unknown15 but a polysaccharide vaccine is unlikely to be efficacious in early childhood due to immaturity of the splenic marginal zone which appears to be required for T-cell independent responses.120 The Vi polysaccharide vaccine is further limited by the short duration of efficacy of 2e3 years.121,122 For these reasons introduction into the Expanded Program on Immunisation (EPI) childhood schedule alongside other routine vaccines during infancy would therefore be of limited if any value. Nevertheless, there are currently (2012) more than ten companies producing different versions of Vi polysaccharide vaccines.

Novel vaccine strategies The development of novel vaccines has been facilitated by scientific advances allowing precise manipulation of bacteria to introduce attenuating mutations.123 Approaches to strain attenuation, including mutations in biochemical pathways, heat shock proteins, regulatory genes and putative virulence genes have all been tried. The availability of the complete genome of strain CT 18124 and subsequently of Ty2125 has facilitated this approach. Despite these advances,

S44 the lack of an animal model in which to assess potential vaccines has hampered efforts to select the best candidates.

Novel oral vaccines Oral vaccination offers the opportunity to induce gut mucosal immunity as well being a logistically easier way to deliver large scale vaccination programmes. Experience with the Ty21a vaccine has demonstrated that it is exceptionally well tolerated and easy to administer, but its application is limited by its moderate efficacy and formulation in capsules. Efforts to increase the immunogenicity of oral typhoid vaccines without compromising safety and tolerability have resulted in several novel oral, singledose vaccines which are currently being evaluated in clinical trials. These include S. Typhi-derived strains M01ZH09, CVD 909 and Ty800. The live, attenuated vaccine M01ZH09, based on the parent Ty2 strain, contains two independently attenuating gene deletions. One, a mutation of the aroC gene, prevents synthesis of aromatic amino acids required for bacterial growth. The second mutation is in the ssaV gene, causing structural abnormality in the specialised type III secretion system encoded by SPI-2 (Salmonella pathogenicity island2).126 Evidence from the mouse model of typhoid infection using S. Typhimurium suggests that the absence of this secretion system leads to an inability of S. Typhi to survive within macrophages.127 Intracellular survival is critical for the systemic spread of S. Typhi, which is thus inhibited by the ssaV mutation.128 In trials of this vaccine to date, systemic infection with the vaccine strain has not been observed.126,129 The safety and immunogenicity of M01ZH09 has been studied in trials in the US, UK and Vietnam. The vaccine is given as a single oral dose and has been well tolerated at all tested doses with mild, self-limiting, gastrointestinal side effects being the most commonly reported adverse event.129 Immunogenicity studies have shown both IgA and IgG responses to the lipopolysaccharide (LPS) of the vaccine.126,130 Immunogenicity and acceptability has been shown in children aged 5e14 years during field trials in Vietnam.130 Phase III trials of M01ZH09 are required to move this vaccine forwards to licensure. In the absence of established correlates of protection however, they would need to be large and of sufficient duration to demonstrate a significant reduction in the local incidence of typhoid fever making them prohibitively costly. The vaccine strain CVD 909 is the latest in a developmental process that commenced with prototype vaccine CVD 908, and progressed via the CVD 908-htrA strain. Strain CVD 908 contains a double attenuation in two genes, aroC and aroD, that rendered it nutritionally auxotrophic for aromatic compounds that are in limited concentrations in human tissue.131 However, although immunogenic,131 silent vaccinaemias occurred in half of the volunteers who received the lower test dose of 5  107 cfu and all of the volunteers who ingested 5  108 cfu of the vaccine strain.123 Although it does not necessarily prevent licensure, the consequent requirement for enhanced prelicensure safety data was felt to be prohibitive and efforts moved towards further attenuation to prevent

C.S. Waddington et al. vaccinaemia. This was achieved by deletion of the htrA gene coding for a heat shock protein that facilitates survival and replication in the host.132 Addition of the htrA mutation to the CVD 908 strain did not decrease immunogenicity (compared with CVD 908) and importantly resulted in disappearance of vaccinaemia.133,134 The intervening development and licensure of the Vi polysaccharide vaccine had demonstrated the protective efficacy of anti-Vi antibodies.113,135 In an attempt to enhance the immunogenicity, CVD 908-htrA was further modified to constitutively express the Vi antigen, forming CVD 909.136 This approach was partially successful, with the majority of volunteers given the highest dose of the vaccine developing an antibody secreting cell response to the Vi antigen.137 More recently, the demonstration that CVD 909 induced anti Vi responses that could be boosted by later administration of the Vi polysaccharide vaccine provided evidence of a memory B cell response to the oral vaccine.138 A further approach to attenuation was used in the development of Ty800 vaccine. The phoP/phoQ regulator region of the Ty2 parent strain was deleted, altering the transcription of multiple virulence properties regulated by this region.139 In dose finding trials, a single dose of this vaccine was shown to be well tolerated and immunogenic.140 Unfortunately, the development of this promising vaccine did not progress beyond phase 1 human clinical studies for commercial reasons.

Novel parenteral vaccines Conjugation of the Vi polysaccharide to a carrier protein to form a protein-polysaccharide conjugate vaccine offers many potential advantages over pure polysaccharide vaccine. Similar to other vaccines where polysaccharides are conjugated to carrier proteins, a Vi-conjugate vaccine induces a T-cell dependent response, with antibody affinity maturation, isotype switching and immunological memory that can be boosted119,120 and protection in infants.120 Examination of responses to pure Vi polysaccharide and Vi conjugate vaccines in mice has also suggested that conjugate vaccines may avoid the hypo-responsiveness observed with plain polysaccharide vaccines in which immune responses to subsequent doses are attenuated.141 The successful conjugation of Vi to a protein carrier was initially described in 1987.142 Conjugation to a nontoxic, recombinant protein (Pseudomonas aeruginosa exotoxin A, rEPA) elicited strong antibody responses that persisted for at least 26 weeks in human volunteers.143 Subsequent field trials of this vaccine in endemic Vietnam showed immunogenicity in children as young as two years of age, and also demonstrated a booster response on repeat administration.144 In an efficacy trial, two doses of the ViPSeprotein conjugate vaccine administered 6 weeks apart were safe, immunogenic and had a protective efficacy of 91.5% in children aged 2e5 years over 27 months’ follow-up.145 Following on from this ground work, a number of manufacturers (information about at least 7 products could be identified in 2012) are pursuing development of Viconjugate vaccines using a variety of protein carriers.146,147 Although one conjugate vaccine is already licensed in India

Enteric fever (but has not been prequalified by the World Health Organisation to date), the majority of these new generation vaccines will complete licensure trials in the next few years.148 The identification of Vi negative strains from patients with typhoid fever149,150 has raised concerns about the immunoprotection induced by vaccines inducing protection directed against the Vi antigen.151 Widespread use of such vaccines might exert selection pressure favouring Vi negative strains, which might then limit their effectiveness. To-date there are no data to support this hypothesis, although there has not been widespread use of Vi vaccines to test it.120 Another recently described approach to vaccine development is the use of a lipopolysaccharideeconjugate vaccine152 which is currently in pre-clinical trials.

Vaccines against paratyphoid fever Setting aside the limited cross-protection provided by Ty21a against S. Paratyphi B,153 no paratyphoid vaccine currently exists. In order to address this need, the approaches that have been used in development of typhoid vaccines are being applied to paratyphoid vaccines. A vaccine consisting of the O-specific polysaccharide of S. Paratyphi A conjugated to tetanus toxoid was found to be safe and immunogenic in adults and children over 2 years, but unexpectedly failed to elicit a booster response.154 A bivalent conjugate vaccine containing both the Vi polysaccharide of S. Typhi and the O-polysaccharide of S. Paratyphi A independently conjugated to CRM197 is under active development.155 Specific mutations in S. Paratyphi A have been used to engineer CVD 1902, a live, mucosal, attenuated vaccine that was immunogenic in animals156 and is now in early human trials. A further attenuated vaccine has been developed by inserting mutations into the same genes (phoPQ) as were used for the attenuation of Ty800, and this has shown promise in animal testing.157

Controlled human infection studies with Salmonella Typhi Animal models of typhoid fever are limited in their applicability to human infection as S. Typhi is a human restricted pathogen. In order to assess typhoid fever vaccines quantitatively,158 controlled human infection experiments with S. Typhi were conducted at the University of Maryland from the 1950s to the 1970s.119 As well as permitting the investigation of typhoid vaccines, these investigations allowed insights into the disease and its pathogenesis to be gained.159,160 Since that time, controlled human infection models of other enteric pathogens have been developed and have demonstrated the considerable scientific value of such experiments. The use of human challenge experiments for the appraisal of candidate typhoid vaccines goes back as far as the first ever typhoid vaccine. A whole cell vaccine, developed by Wright in 1896,161 was administered to two medical officers, one of whom underwent subsequent challenge with an unknown dose of S. Typhi.162 Illness did not occur, and this was taken as evidence of successful vaccination!

S45 Having established the number of organisms required to cause a given rate of infection,163 the use of a controlled human infection model in vaccine appraisal was evaluated by using two killed whole cell vaccines for which field efficacy data were available. Following oral challenge with 105 bacteria, a dose known to induce disease in 25% of nonvaccinated controls (ID25 dose), the protective efficacy of both vaccines corresponded with that observed in field trials, demonstrating the applicability of the model. Interestingly, a challenge dose that exceeded the ID25 overwhelmed the protection offered by the vaccines. The model was also used in the initial appraisal of the Ty21a vaccine. Ty21a was shown to protect 87% of volunteers, to be safe and well tolerated and to decrease excretion of virulent S. Typhi after challenge.103 These data provided the impetus to undertake field trials of the vaccine and ultimately contributed to the licensure and successful use of this vaccine.

Establishing a 21st century challenge model Novel, affordable typhoid vaccines with improved efficacy over existing vaccines that can be used in infants are desperately needed. Vaccine development is a long, costly and challenging process with many potential vaccines failing to progress on the path to licensure.164 The poor understanding of typhoid immunobiology, and, in particular, the absence of a correlate of protection for typhoid fever that could be used in efficacy trials was highlighted as a rate-limiting factor in the slow development of Ty21a.164 Although it is nearly 20 years since both Ty21 and the Vi polysaccharide vaccines were licensed in the United States, our understanding of typhoid immunobiology remains poor, correlates of protection still do not exist and as a result development of novel approaches to typhoid vaccine development is hampered. It is hoped that re-establishing a controlled human infection model of typhoid fever, and later paratyphoid fever, could accelerate development of improved vaccines, diagnostics and treatment. We have developed a controlled human infection model of typhoid at The University of Oxford to study the early inflammatory and immune responses to S. Typhi infection and to identify novel diagnostic markers. By combining vaccine efficacy data with immune-response data from volunteers who have been challenged with S. Typhi it may be possible to identify correlates of protection for typhoid fever, thus facilitating progress to phase III trials and studies in new populations.

Conclusion Infections caused by S. Typhi and S. Paratyphi cause a considerable world-wide burden of disease. Clinical diagnosis is difficult and reliable diagnostic tests are not available. Moderately efficacious vaccines against typhoid fever are available today but have not been widely deployed and none are suitable for young children. Novel vaccines are needed, and there are several promising strategies on the horizon. However, as a human restricted pathogen, clinically relevant models for appraising these vaccines and improving our understanding of the disease are lacking. Previously, a controlled human infection model

S46 of typhoid fever was established and provided much of the current understanding of disease pathology as well as providing support for the Ty21a vaccine to be developed. A 21st century controlled human infection model has been developed to further understanding of enteric fever and its prevention and we hope it will provide important data to advance the control of this important disease.

Conflict of interest The authors have no conflict of interest to report.

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