Diarrhoeal Diseases (Updated February 2009)
Rotavirus
Introduction
Rotaviruses (RV) are the leading cause of severe diarrhoeal disease and dehydration in infants and children under the age of 5 worldwide [131] . The global situation recently changed with the advent of new oral RV vaccines [132] . Three oral RV vaccines are currently licensed, a human monovalent live attenuated RV strain, RotarixTM, a pentavalent live bovine-human reassortant vaccine, RotaTeqTM, and a lamb-derived monovalent live attenuated strain, LLR, which is only being used in China. Several countries have introduced the RotarixTM and RotateqTM vaccines into routine immunization programmes, which hopefully will greatly reduce the burden of gastroenteritis and dehydration worldwide and have a strong impact on infantile diarrhoea mortality in developing countries [133] . A few data on the effectiveness of these vaccines in poor African or Asian countries have recently been made available, but concerns remain regarding the potential effectiveness of any oral live vaccine in these settings in view of prevalence of competing intestinal flora in children, occurrence of mixed infections, high levels of maternally transmitted antibodies and micronutrient malnutrition.
Meanwhile, different, new vaccine approaches such as non-replicating virus-like particles (VLP) vaccines and other routes of administration are being tested in animal models and will soon be evaluated in humans [134].
Disease Burden
Rotavirus is currently by far the most common cause of severe diarrhoea in infants and young children worldwide and of diarrhoeal deaths in developing countries [135] with a distinct winter seasonality in temperate climates and year-round exposure in tropical countries. Virtually all children are infected by the time they reach 2 to 3 years of age. Most symptomatic episodes occur between 3 months and 2 years of age with a peak incidence between 7 and 15 months. Symptoms include watery diarrhoea, nausea, vomiting, abdominal pain and dehydration. Outbreaks of RV gastroenteritis in day-care centers and hospitals can spread rapidly among nonimmune children, presumably through person-to-person contacts, airborne droplets, or contact with contaminated toys [136] . Children from low socioeconomic background and low birth weight infants have an increased risk for hospitalization [137].
RV infection can also occur in adults [138] , especially in institutionalized or hospitalized elderly patients [139] . Both symptomatic and asymptomatic patients shed RV in their stools for 7-10 days, but shedding can happen to last for several weeks. The virus is highly resistant in the environment and can survive for months in stools at room temperature [140].
Worldwide, RV has been estimated to account for almost 40% of all cases of severe infant diarrhoea [141] [142] , which translates into 527 000 deaths each year (range: 475 000-580 000), mostly in children under age 2 [83].[135] Mortality still is the greatest in south and south-eastern Asia and sub-Saharan Africa, with almost 100,000 deaths each year in India alone and more than 200 000 in African countries [143] . The Asian Rotavirus Surveillance Network, which involves 14 countries working in collaboration with the WHO, PATH and the Centers for Disease Control and Prevention (CDC) in Atlanta, GA (USA) estimated that 73% of hospital admissions of children for diarrhoea in South Korea were RV positive, 58% in Japan, 55% in Vietnam, 53% in Myanmar, 46% in China, 43% in Thailand and 30% in HongKong.
Before the introduction of vaccination, RV gastroenteritis was estimated in the USA to account for over 50 000 hospitalizations, 200 000 emergency department visits, 410 000 physician office visits, and 20 to 40 deaths per year. In 2006 in the region of the Americas there were more than 10 million episodes of RV diarrhoea requiring domiciliary visits, 2 million requiring a clinic consultation, and 75,000 requiring hospitalization, leading to considerable medical costs (more than US$ 17 million in Mexico alone). In Asia, universal RV immunization would avert about 110 000 deaths, 1.4 million hospitalizations and 7.7 million outpatient visits [83].
Virology
Rotaviruses are 70 nm icosahedral, non-enveloped, double-stranded RNA viruses that belong to the family Reoviridae. The virus is characterized by its three-layer capsid, an outer and an inner capsid and an internal shell that surrounds the 11-segment double-stranded RNA genome. The outer capsid is made of two proteins, VP4, also named "P protein", and VP7, also known as the "G protein", which define the "P" and "G" serotypes of the virus, respectively. Both are key neutralization determinants on the surface of the virion. The inner capsid is made of the VP6 protein, the most abundant and immunogenic protein in the virion. Anti-VP6 antibodies do not neutralize virus infectivity but VP6-specific IgAs appear to confer protection in vivo, perhaps through inhibition of virus transcytosis through the intestinal epithelium barrier [144].
When mixed infections with distinct RV strains occur, the individual genomic RNA segments can reassort independently, producing progeny "reassortant particles" of mixed parentage, which could theoretically lead to the emergence of up to 110 different G and P combinations. In fact, G serotypes G1-G4 and G9, and P genotypes P[4] and P[8] are predominant worldwide, causing aver 90% infections in industrialized countries and about 68% infections in South American and Asian countries [145] . P[8]-G1 is the globally predominant strain, followed by P[8]-G3, P[4]-G2, and P[8]-G4 [146] . G9 strains have emerged in the early 2000s and have become predominant in some regions of the world, including Europe, Thailand and parts of Eastern Asia. Less usual strains may also be found, such as P[6]-G8 in Africa, P[8]-G5 in Brazil and novel P[11]-G10 and P[6]-G12 in India [147].
Rotavirus surveillance system networks have been constituted with the collaboration of the CDC in Atlanta (USA) and WHO to estimate the hospital-based disease burden of RV gastroenteritis in children less than five years of age, and to constantly update the frequency and characteristics of circulating strains. This aspect is of importance for the development of RV vaccines and for studying the possible vaccine selective pressure leading to emergence of new strains in the vaccinated populations.
Vaccine
Natural RV infection protects partially against reinfections, a first episode in newborns or young infants attenuating the severity of diarrhoea during subsequent episodes. Reinfection seems to broaden and boost natural immunity. Usually, complete protection against severe gastroenteritis is acquired after the second infection. Therefore, even if vaccination early in life may not prevent all subsequent disease episodes, it should prevent most cases of severe RV disease and their complications such as dehydration, physician visit, hospitalizations and deaths [134]. Because RV remains the most common cause of severe diarrhoea in children in all regions of the world, an RV vaccine will have universal application as part of childhood vaccination programmes [148].
Live attenuated RV strains
The first RV vaccines to be tested in humans were the live bovine strains RIT4237 (P[1]-G6) and WC3 (P[5]-G6), and the live simian strain RRV (P[3]-G3), which are attenuated for humans and could be administered by the oral route. The three strains induced neutralizing antibodies in a majority of infants but showed inconstant capacity to protect against RV disease.
In China, a lamb-derived monovalent (P[12]-G10) live-attenuated, 3-dose oral vaccine, was developed by the Lanzhou Institute of Biomedical Products and is used in the private sector. The vaccine is reported to induce neutralizing antibody responses in 60% of vaccinees but its efficacy is not precisely known since it was not tested against placebo in a controlled Phase III trial [135].
A human P[8]-G1 RV strain, RIX 4414, which was isolated from the stools of a sick 15-month old boy in the USA, was attenuated by multiple passages in cell culture, plaque-purified and passaged again in Vero cells. The strain was developed as a 2-dose monovalent oral vaccine by AVANT Immunotherapeutics then licensed to GlaxoSmithKline Biologicals. The vaccine (RotarixTM) showed 70%-85% protective efficacy against severe disease, including that due non-G1 serotypes [149] [150] . It now has been tested in more than 60 countries in Latin America, Africa, Asia and Europe. A large, multicentered safety trial on 63 225 infants between 6 and 14 weeks of age in Latin America and Finland confirmed the initial safety data and indicated no increased attributable risk of intussusceptions (IS) in the high-risk period up to 30 days post any dose [151] . The vaccine was first licensed in 2004 in Mexico and the Dominican Republic and has now been licensed in many countries worldwide. It also has been prequalified by WHO for procurement by UNICEF and the UN Vaccine Fund. Additional Phase IIb and III trials are in progress in South Africa, Malawi and Bangladesh to determine if the vaccine will work well in children from poor settings in developing countries, if it can be administered with the oral polio vaccine without interference, and whether it can safely be administered to HIV positive infants. Final results are due in 2009.
Another human RV strain, RV3 (P2[6]-G3), isolated from newborns at the Royal Hospital in Melbourne, Australia [152] is also developed as a candidate live oral vaccine. A small Phase II study with three doses of 105 pfu of the vaccine indicated relatively low immunogenicity in infants as measured by serum IgA levels [153] . However, the vaccine recipients who developed an immune response were protected against clinical disease in the following year. Strategies to increase the potency of the vaccine are under study with a vaccine producer in Indonesia.
Live reassortant RV strains
Efforts also were made to develop human-animal reassortant RV strains containing the VP7 or VP4 RNA segment from a human RV strain to provide the required antigenicity and the other 10 RNA segments from a simian or a bovine strain to provide the attenuated phenotype [154] [155].
A tetravalent rhesus-human reassortant RV vaccine, RRV-TV, was initially developed at the NIH, Bethesda, using the simian RRV strain (G3) mixed with three human-simian reassortant strains of G types 1, 2, and 4, respectively. The vaccine (RotaShieldTM, Wyeth-Lederle Vaccines, USA) was shown to provide 48-68% protection against any RV disease and 64-91% protection against severe disease [156] . It was introduced in August 1998 on the market in the USA and administered in a three dose schedule to over 600 000 infants within the following year, until an unexpected adverse event, intussusception (IS), was found to occur in a significant number of cases within two weeks after administration of the first two doses of vaccine, leading to its eventual withdrawal [157] . The risk of IS, initially targeted at 1 in 2500 children immunized, has now been reassessed as 1 in 10 000. Its occurrence led to a very thorough safety assessment of the following generation of live oral RV vaccines (viz RotarixTM and RotaTeqTM), with sample sizes in excess of 60 000 subjects. The original RotashieldTM vaccine has now been licensed to a biotech company, BIOVIRx Inc., USA.
A pentavalent human-bovine reassortant vaccine, RotaTeqTM, was prepared by Merck Research Co., Pennsylvania, by reassortment between the naturally attenuated bovine RV strain WC3 and five different human RV strains of serotypes G1, G2, G3, G4 and P[8], respectively. The live-attenuated, 3-dose oral vaccine, was tested in a large safety and efficacy trial in Finland and the USA on more than 70 000 children who were carefully monitored for 2 weeks after each immunization for risks of IS. The vaccine was found to be totally safe and to elicit 74% protection against any G1-G4 RV gastroenteritis through the first RV season after vaccination [158] . Vaccination reduced doctor visits for RV diarrhoea by 86% and hospitalizations and emergency department visits by 94.5%. The vaccine was shown not to interfere with the immunogenicity of a combined Hib, DTP, HepB, conjugated pneumococcal and inactivated polio vaccine, nor with concomitant administration of the oral polio vaccine [159] . RotaTeqTM was licensed in February 2006 in the USA and subsequently in many countries worldwide. It officially was recommended for the routine immunization of children in the USA after active surveillance showed only three cases of IS among more than 100 000 vaccinated infants. It also has been included into national vaccination programs in several countries. A Phase III trial is ongoing in African countries (Mali, Ghana, and Kenya). Results are expected end of 2009.
An alternative multivalent bovine-human reassortant oral vaccine was developed by the National Institute of Allergy and Infectious Diseases (NIAID, NIH, Bethesda), based on the attenuated bovine strain UK reassorted with the five most common RV serotypes in humans, G1-G4, G8 and G9 [156] . Phase II data showed a good immune response and no adverse interference with concomitantly administered childhood vaccines. A non-exclusive license for production of the vaccine has been granted to vaccine producers in Brazil, China and India.
Finally, a naturally occurring human-bovine, neonate-derived RV strain, 116E (P[10]-G9), which was isolated from a nosocomial outbreak of asymptomatic infection in New-Delhi, is under development by Bharat Biotech Ltd in India [148] [160] . A similar strain, I321 (P[11]-G10), was found not to be immunogenic [161].
Other RV vaccine approaches
New RV vaccine approaches include an inactivated virus vaccine [162] , DNA vaccines [163] , a VP6 subunit vaccine [164] [165] and virus-like particles (VLPs) expressed in a baculovirus system [166] [167] [168] Depending on the number of viral proteins expressed, the complexity of the VLPs can vary from mono-layered (VP2-VLPs) to double-layered (VP2/6 VLPs) or triple- layered VLPs (VP2/6/7/4 VLPs).
These candidate vaccines have been found to be immunogenic in mice and rabbits after administration by the parenteral, transcutaneous, oral, nasal or rectal routes. Whether they will be further developed will depend on the outcome of the current live oral RV vaccines, which are facing several key questions in regard with cost, efficacy in developing countries and safety [169] [170] Phase III trials of the RotarixTM and RotaTeqTM vaccines have been initiated by PATH in partnership with GSK in Malawi and South Africa and with Merck in Kenya, Mali, Ghana, Vietnam and Bangladesh. Even if the two live oral RV vaccines prove to be efficacious in these settings, however, their current price makes them unaffordable to poor countries [171] and new financing mechanisms will have to be set up for vaccination to be implemented on a routine basis in these countries.
Another matter to watch carefully will be that of cross-protection against the full range of RV strains, including serotype G9, which is becoming increasingly important across Asia, and G8, which is gaining prevalence across Africa.
The benefit of vaccination can however be observed in the recent CDC report [172] , which indicates a more than 50% reduction in seasonal incidence of RV during 2007-2008 in the USA, coinciding with increased use of RV vaccine. During the first 18 weeks of 2008, only 6% of samples tested positive for RV, compared to 51% in 2006 and 54% in 2007 over the same period.