New breath for rotavirus vaccines

Drug Discovery Today: Therapeutic Strategies

Vol. 3, No. 2 2006

Editors-in-Chief Raymond Baker – formerly University of Southampton, UK and Merck Sharp & Dohme, UK Eliot Ohlstein – GlaxoSmithKline, USA DRUG DISCOVERY

TODAY THERAPEUTIC

STRATEGIES

Infectious diseases

New breath for rotavirus vaccines Umesh D. Parashar*, Joseph S. Bresee, Marc-Alain Widdowson, Jon R. Gentsch Viral Gastroenteritis Section, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30333, USA

New vaccines against rotavirus, the leading cause of severe childhood gastroenteritis worldwide, are on the threshold of introduction into immunization programs in affluent nations. A reduction of the health burden

Section Editors: Gary Woodnutt – CovX, San Diego, USA Paul-Henri Lambert – Centre of Vaccinology, University of Geneva, Switzerland

and costs of severe rotavirus disease in these countries should be evident within 2–3 years of vaccine introduction. However, realizing the full potential of these vaccines will require demonstration of their efficacy

subsequent severe disease [7], and immunization is expected to mimic this protection from natural infection. This article reviews the main strategies (Table 1) used to develop candidate live, oral rotavirus vaccines.

and ensuring their affordability in the poorest populations where rotavirus kills >500,000 children annually. Introduction The need for effective interventions against rotavirus gastroenteritis is evident from the tremendous global health burden of this disease (Fig. 1) [1,2]. In developing countries, rotavirus accounts for approximately one-quarter of the 2 million annual deaths from childhood diarrhea or 5% of all childhood deaths (Fig. 2). Whereas rotavirus-associated deaths are uncommon among children in industrialized nations [3], this pathogen accounts for 35–50% of episodes of childhood gastroenteritis episodes requiring hospitalization or physician visit, with considerable health and economic impact [4,5]. No specific drug for treatment of rotavirus is available. For several reasons, vaccines offer the greatest promise for prevention of severe rotavirus disease. First, rotavirus infects >90% of children in both developing and industrialized countries by age 5 [6], indicating that further improvements in water or hygiene are unlikely to have a substantial impact. Second, access to and use of oral hydration therapy for diarrhea remains problematic in many parts of the world. Finally, studies indicate that natural rotavirus infection protects against *Corresponding author: U.D. Parashar ([email protected]) 1740-6773/$ .Published by Elsevier B.V.

DOI: 10.1016/j.ddstr.2006.06.005

Properties of rotavirus relevant to vaccine development Rotaviruses, members of the family Reoviridae, are characterized by three concentric shells (outer capsid, inner capsid and core) that surround 11 segments of double-stranded RNA (Fig. 3) [6]. Each RNA segment encodes a protein and two proteins that form the outer capsid, VP7, a glycoprotein (Gprotein) and VP4, a protease-cleaved protein (P-protein), represent both the targets for the immune system to mount a neutralizing antibody response and are the key antigens used to characterize strains. Although theoretically 140 different G and P combinations could result by reassortment during mixed infections in vivo from the 10 different VP7 (Gserotypes) and 14 different P-serotypes that are known to exist, only five strains (P[8] G1; P[4] G2; P[8] G3; P[8] G4; P[8] G9) are globally prevalent and are the primary targets for vaccine development (Fig. 4) [8,9]. The ability of rotaviruses to readily undergo genetic reassortment (see Glossary) during co-infection to yield mixed progeny with gene segments derived from each parent strain, has also been utilized to develop reassortant vaccines in vitro. Children can be infected with rotavirus several times in the first few years of life, but the first infections are generally symptomatic and most severe, with each subsequent infection conferring greater protection against both re-infection 159

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Strategy 1 – non-human rotavirus strains Glossary Genetic reassortment: exchange of genomic RNA segments between two rotavirus strains (e.g., serotype P[8],G1 and P[4],G2 strains) during co-infection of a single cell in vivo or in vitro with both strains such that the resultant progeny viruses contain RNA segments derived from both parent viruses. An example would be a progeny virus of genotype P[8], G2. Intussusception: the slipping of a length of intestine into an adjacent portion usually producing obstruction. Quadrivalent: composed of four homologous virus strains that differ only in the VP7 gene that specifies G serotype. Plaque-cloned: a virus strain that has been genetically purified from a genetically heterogeneous population by isolating individual plaques from a standard plaque assay of the strain. In the procedure, cell cultures are infected with serial 10- fold dilutions of the virus culture to be plaque-cloned and then the infected cells overlaid with an agarose-media mixture to restrict the spread of the virus. At higher dilutions, one or a few foci originating from infection of a single cell by a single infectious virus particle can be visualized using a viable stain and then isolated by aspiration. The isolated plaque is usually re purified several more times by the same procedure and then expanded in culture.

and severe disease [7]. The mechanisms of protection against rotavirus disease are unclear. Whereas the presence of rotavirus-specific IgA at the intestinal mucosal surface and in serum correlates with protection in studies of natural rotavirus infection, this correlation has not been consistently observed in vaccine trials so clinical trials of efficacy are the only reliable means to assess candidate vaccines [10–12]. Following primary infection with rotavirus, children develop neutralizing antibodies in serum directed primarily against the G serotype of the infecting (homotypic) strain. The presence of high enough levels of these antibodies correlates with protection to the infecting strain. Repeat rotavirus infections elicit both a homotypic and heterotypic antibody response. Thus, it remains unclear whether effective rotavirus vaccines will need to contain G and P types of all prevalent strains or whether repeat doses of a single strain vaccine will induce sufficient crossprotection against other strains.

The first generation of rotavirus vaccines was based on the approach used by Jenner to develop smallpox vaccines, in which a related, live attenuated rotavirus strain from a nonhuman host was used as the antigen. Because of observations that human and animal rotaviruses share a common group antigen and that children with rotavirus diarrhea develop an immune response to human as well as certain bovine, simian and murine rotaviruses, it was hoped that infection of children with a naturally attenuated non-human rotavirus vaccine strain would also confer cross-protection against human rotaviruses. Studies in animal models also demonstrated the potential of immunization with animal rotaviruses to protect against human rotavirus infection.

Bovine rotavirus vaccines RIT4237 and WC3 The first ‘Jennerian’ vaccines were developed by using the bovine rotavirus strains RIT4237 (P[6] G6) and WC3 (P[7] G6). Both vaccines showed promising results in initial efficacy trials (e.g. efficacy of RIT4237 was 55–62% against any rotavirus diarrhea and 80–88% efficacy against severe disease in two Finnish trials). However, considerably lower efficacy was observed in trials conducted in developing countries such as Rwanda, Gambia, Peru and the Central African Republic, and both vaccines were withdrawn from further development [13–19].

Rhesus rotavirus vaccine MMU18006 The next ‘Jennerian’ vaccine was developed by using the rhesus rotavirus strain MMU18006 (P[5] G3), which shares neutralization specificity with human rotavirus G3 strains. Like RIT4237 and WC3, the protective efficacy of MMU18006 in field trials was inconsistent [19,20], and further development was not pursued.

Lanzhou lamb rotavirus vaccine (Lanzhou Institute, China) Because of their inconsistent performance,1 monovalent nonhuman rotavirus strain vaccines have been mostly abandoned, with exception of the Lanzhou lamb rotavirus (LLR) vaccine. LLR is a monovalent, lamb strain (P[12] G10) vaccine produced by the Lanzhou Institute and licensed in China in 2000. Although LLR is available and is currently being used in some parts of China, several factors including the lack of a proper clinical trial have precluded its widespread use in China or elsewhere.

Figure 1. Estimated global burden of rotavirus disease [1]. This figure shows the tremendous overall morbidity due to rotavirus worldwide. By 5 years of age: all children will be infected with rotavirus, 1 in 5 will visit on outpatient clinic, 1 in 60 will be hospitalized and 1 in 293 will die.

Strategy 2 – reassortant animal–human rotavirus strains Following the inconsistent performance of monovalent non-human rotavirus vaccines, emphasis shifted on development of multivalent animal–human reassortant vaccines 1

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Figure 2. Estimated global distributions of deaths from rotavirus disease. One dot = 1000 deaths [1]. This figure illustrates the high mortality due to rotavirus, which is particularly concentrated in the poorest regions of the world: in south Asia and subSaharan Africa.

because of certain observations. First, the greatest efficacy of the monovalent MMU18006 vaccine was observed in a Venezuelan trial, in which the circulating strain of rotavirus in the community (G3) was the same serotype as the vaccine strain, suggesting that serotype-specific immunity can be required for maximum protection. Analogous conclusions were reached on the importance of serotype-specific immunity in some studies of natural rotavirus infection. Similar observations made in challenge studies of cross-protection among vaccinated animals initiated the development of reassortant vaccines that had the attenuation properties of the animal (rhesus or bovine) strains and single genes encoding the outer capsid proteins of the common human strain [21].

Quadrivalent rhesus rotavirus-based rhesus-human reassortant (RotaShield1, BIOVIRx,1 USA) RotaShield1 is a quadrivalent (see Glossary) human-rhesus reassortant vaccine that includes rhesus rotavirus (RRV) of G3 specificity, with three reassortants, each of which possesses the VP7 gene from the human rotavirus serotype 1, 2 or 4 and the other 10 genes from RRV [22]. Seven large efficacy trials were conducted [23], which demonstrated consistent estimates of 50–60% protection against all cases of rotavirus diarrhea and 70–100% protection against severe rotavirus disease. RotaShield1 showed efficacy against multiple strains that persisted for up to 3 years. RotaShield1 was associated with short-lived episodes of fever 3–5 days following the first vaccination. Five intussusception events were reported among approximately 10,000 vaccine recipients in prelicensure trials – four of these events occurred within 3

weeks after administration of the second or third dose [24]. Three of the four events occurred in a subset of <2000 infants who were given experimental vaccine formulations that were never marketed. Intussusception was listed in the package insert as an adverse event that was noted in prelicensure trials but was not statistically associated with vaccination. RotaShield1 was licensed by the Food and Drug Administration2 in August 1998, went immediately into the routine schedule of childhood immunization in the United States [23], and was administered to >600,000 infants in the first 9 months of the program. In June 1999, following administration of approximately 1.2 million doses of RotaShield1 to 600,000 infants, vaccination was suspended because of reports of intussusception (see Glossary) among vaccinated infants. Subsequent studies confirmed an association between RotaShield1 and intussusception, with the greatest risk during 3–14 days after receipt of the first dose of vaccine [25]. The risk of intussusception associated with the first dose of RotaShield1 was estimated to be 1 case per 10,000 vaccine recipients. In October 1999, the recommendation for use of RotaShield1 was withdrawn and subsequently the manufacturer (Wyeth Lederle Vaccines,3 USA) stopped its production. In 2004, the license for RotaShield1 was transferred to BIOVIRx, Inc. (Minneapolis, MN, USA), that is currently re-evaluating the vaccine and its commercial prospects.

2 3

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Strategy

Pros

Cons

Latest developments (including drug therapies in progress and failures)

Who is working on this strategy (include web address)

Refs

Non-human rotavirus strains

Simple approach using virus naturally less virulent for human population

Single strain vaccines with inconsistent efficacy in trials

 Development of most candidates discontinued more than 10 years ago after disappointing clinical trials  Lamb rotavirus vaccine (LLR) licensed in China since 2000 but only being used locally; LLR has not been fully evaluated in an acceptable field trial, so safety and efficacy are not fully known

LLR – Lanzhou Institute, China (web address not available)

n/a

Reassortant animal–human rotavirus strains

Many strains to provide broad coverage against circulating strains

Technically complex manufacturing process

 RotaShield1 – licensed in US in 1998; withdrawn in 1999 because of intussusception; licensed to BIOVIRx in 2004  Rotateq1 – completed successful phase III clinical trial in 2005; licensed by US FDAa and EMEA (European Medicines Agency) in 2006  UK-based vaccine – licensed by the US NIHb for early development to companies in India, China and Brazil

 RotaShield1 – BIOVIRx, USA

[27]

 Rotateq – Merck and Co., USA

Human infant rotavirus strains

Replicates well in the infant gut and does not cause disease because of attenuation by serial passage

Single strain vaccine relies on cross-protection against other strains

Rotarix1 – licensed in >40 countries in Latin America, Asia, and Europe; not currently submitted to US FDA; in clinical trials in developing countries (Bangladesh and South Africa)

GlaxoSmithKline Biologicals, Belgium

[32]

Human neonatal rotavirus strains

Naturally attenuated virus replicates well even in presence of maternal antibodies

Limited data on clinical efficacy of these relatively early stage vaccines

 RV3 vaccine – completed phase II trials; being further developed to increase vaccine titer  116E and I321 vaccines – pilot lots being developed for human studies

116E and I321 vaccines – Bharat Biotech International Limited, India

[33]

a

Food and Drug Administration. National Institutes of Health.

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Table 1. Main strategies used to develop live, oral rotavirus vaccines and current status

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Figure 3. Gene coding assignments and three-dimensional structure of rotavirus particles. Double-stranded RNA segments separated on polyacrylamide gel (left) code for individual proteins, which are localized in the schematic of virus particle (center) or in different protein shells of virus (right). Outer capsid proteins VP4 and VP7 are neutralization antigens (P and G proteins, respectively), which induce neutralizing antibody; protein that makes up intermediate protein shell, VP6, is the subgroup antigen. The 11 RNA separate segments are seen as red lines in the center of the virion and illustrate the capacity of the rotavirus genome to reassort by segment exchange. VP: Viral protein (1–4, 6, 7); NSP: nonstructural protein (1–5) [6].

Figure 4. Distribution of rotavirus strains from a global collection of strains. ‘Others’ includes strains that were not typable [9]. G and P refer to the two main neutralization proteins of which there are several types (Fig. 3). This figure shows that strains with G1–4 or P[8] proteins comprise at least 75% of all strains globally.

Pentavalent WC3-based bovine-human reassortant (Rotateq1 Merck Inc., USA) Rotateq1 is a live,4 oral vaccine that contains five reassortant rotaviruses developed from human and bovine (WC3 [P[7] 4

http://www.merck.com.

G6]) parent rotavirus strains [26]. Four reassortant rotaviruses express one of the G serotype antigens (G1, G2, G3 or G4) from the human rotavirus parent strain and (or the P serotype protein) attachment protein (P[7]) from the bovine rotavirus parent strain. The fifth reassortant virus expresses the attachment protein (P8) from the human rotavirus parent strain and the outer capsid protein G6 from the bovine rotavirus parent strain. Rotateq1 consists of the five human-bovine reassortants suspended in a buffered stabilizer solution that can be directly administered to infants and is delivered in three oral doses beginning at 6 weeks of age. The efficacy and safety of Rotateq1 has been evaluated in the large rotavirus efficacy and safety trial (REST) study of more than 70,000 infants conducted primarily in the United States and Finland [27]. Intussusception was closely monitored during the 42-day postvaccination period and six cases of intussusception were identified among Rotateq1 recipients compared with five cases of intussusception in the placebo group (multiplicity adjusted relative risk: 1.6, 95% CI: 0.4, 6.4). No evidence of clustering of cases of intussusception was observed within a 7- or 14-day window postvaccination. In a subset of about 7000 subjects in whom efficacy was monitored, Rotateq1 was 74% (95% CI: 67%, 79%) efficacious against G1–4 rotavirus gastroenteritis of any severity and 98% (95% CI: 90%, 100%) efficacious against severe G1–4 rotavirus gastroenteritis. Efficacy was observed against all G1–4 serotypes, although relatively few non-G1 rotavirus gastroenteritis cases were reported in the trial. The efficacy of Rotateq1 in the second rotavirus season postvaccination was 63% (95% CI: 44%, 75%) against G1–4 rotavirus www.drugdiscoverytoday.com

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gastroenteritis of any severity and 88% (95% CI: 49%, 99%) against severe G1–4 rotavirus gastroenteritis. Rotateq1 reduced the incidence of office visits by 87% (95% CI: 78%, 92%), emergency room visits by 94% (95% CI: 89%, 97%), and hospitalizations for rotavirus gastroenteritis by 96% (95% CI: 91%, 98%). Efficacy against all gastroenteritis hospitalizations of any etiology was 59% (95% CI: 56%, 65%). Rotateq1 was licensed in the United States and in Europe in 2006, and license applications have been submitted in Europe, and several countries in Latin America and Asia.

Quadrivalent UK-based bovine-human reassortant vaccine (National Institutes of Health, USA) A second bovine-based reassortant vaccine has been developed based on the UK rotavirus,5 a P[7] G6 strain [28]. Reassortants containing 10 genes from the parent UK stain and one gene for each of the four common human VP7 serotypes (G1, G2, G3 and G4) are contained in the vaccine, and reassortants for types G8 and G9 will also be added in the future to this vaccine. In a preliminary trial in Finland, the efficacy of the quadrivalent vaccine was comparable to the RotaShield1 vaccine but caused less fever. This vaccine has been licensed by NIH to vaccine manufacturers in Brazil, China and India for further development.

Strategy 3 – human infant rotavirus strain Rotarix1 (GlaxoSmithKline Biologicals, Belgium6) was developed from a human rotavirus strain, 89-12 (P[8] G1), that was further passaged and plaque-cloned (see Glossary) to yield the vaccine strain, RIX4414 [29]. The vaccine is prepared as a lyophilized powder that is reconstituted with 1 ml of a citrate bicarbonate buffer at the time of administration and delivered as two oral doses, with the first administered at approximately at 2 months of age and the second dose at 4 months of age. In initial studies in Latin America (Mexico, Venezuela, Brazil) and Asia (Singapore), the vaccine conferred an efficacy of 70–85% against any RV diarrhea and 85–93% against severe disease [30,31]. Furthermore, data from the efficacy trial in Latin America demonstrated significant efficacy 83% (95% CI: 40%, 97%) against non-G1 serotypes, which were predominantly P[8] G9 strains. As for the Rotateq1 vaccine, a large trial to evaluate the safety of Rotarix1 with respect to intussusception was conducted in more than 63,000 infants enrolled from 11 countries of Latin America and from Finland [32]. In a subset of 20,000 infants in this trial, Rotarix1 prevented 85% of all severe rotavirus diarrhea and reduced hospitalizations for diarrhea of any cause by 41%. During the 31-day postvaccination window after each dose, six and seven intussusception cases were reported between Rotarix1

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and placebo recipients, respectively (risk difference, 0.32 per 10,000 vaccinees; 95% CI: 2.91, 2.18). Since the launch of Rotarix1 in Mexico in 2004, more than 40 licensed have been granted worldwide and license applications have been filed in 75 other countries. In 2006, Brazil and Panama included for the first time the rotavirus vaccine in their national vaccination calendars.

Strategy 4 – human neonatal rotavirus strains Neonatal rotavirus strains have been pursued as vaccine candidates because they often infect newborns at a high prevalence without causing clinical illness and this neonatal infection protects against severe rotavirus disease in early childhood. Two strains isolated in India during outbreaks of asymptomatic infection in newborn nurseries, 116E (P[11] G9) and I321 (P[11] G10), are being pursued as candidate vaccines [33]. Both strains are natural bovine-human rotavirus reassortants that are being developed by an Indian company, Bharat Biotech International Limited,7 Hyderabad, India, and will undergo clinical testing over the next 4–7 years. RV3, a P[6] G3 strain, was isolated from newborns at the Children’s Hospital in Melbourne.8 A trial of three doses of vaccine induced immune responses in 54% of infants and those who developed an immune response were protected from rotavirus disease [34], leading the developers (in conjunction with BioPharma, Bandung,9 Indonesia) to work on efforts to increase the titer of this vaccine and return to clinical trials. M37, a P[6] G1 newborn strain isolated in Venezuela was developed as a vaccine candidate but was abandoned after poor results in initial efficacy trials.

Conclusions Despite the abrupt and unanticipated setback of the withdrawal of the first licensed rotavirus vaccine, RotaShield1, two new safe and effective vaccines for prevention of the tremendous global burden of rotavirus disease might soon be available. However, several issues remain to be addressed before the promise of rotavirus vaccines can be fully realized (Outstanding issues). A fundamental unanswered question is whether these live oral vaccines will perform as well in developing countries as they have in middle- and high-income countries where they have been tested to date. Many factors in developing countries – younger age at infection, potentially larger inoculum of infection, breast-feeding practices, presence of unusual rotavirus strains, interference by other enteropathogens, poorer nutritional status of children, and comorbid conditions such as HIV – could adversely affect the performance of 7

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http://www.bharatbiotech.com. http://www.rch.org.au. http://www.biofarma.co.id.

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rotavirus vaccines [35]. Moreover, efficacy of these vaccines has not been studied when co-administered with oral poliovirus vaccine. Studies to address many of these issues are currently being undertaken. The legacy of intussusception that led to the demise of RotaShield1 could be a barrier to the adoption of new rotavirus vaccines by health care providers and parents. Even though testing of both the Rotarix1 and Rotateq1 in more than 130,000 total infants provides reassuring data that these vaccines might not be associated with intussusception, the clouds of uncertainty over the safety of these vaccines will remain to an extent until additional postlicensure surveillance data become available. The issue of affordability will be a key consideration for adoption of these new vaccines by the poorest countries where the burden of rotavirus disease is the greatest. The Global Alliance for Vaccines and Immunizations10 has identified Streptococcus pneumonia and rotavirus vaccines as priorities for rapid development and introduction and has provided resources to develop the evidence base which countries could use to make an informed decision concerning introduction of these vaccine. Although these data will be useful, ultimately, however, it is probable that the cost of vaccines will play a key role in their adoption and donor support will be needed for the poorest developing countries to sustain an immunization program.

Outstanding issues  Efficacy of rotavirus vaccines in developing country settings.  Safety with respect to intussusception during routine programmatic use.  Impact of intussusception associated with RotaShield1 on uptake of new vaccines.  Affordability of vaccine for poor countries.

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