http://informahealthcare.com/mby ISSN: 1040-841X (print), 1549-7828 (electronic) Crit Rev Microbiol, Early Online: 1–11 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/1040841X.2014.962479

REVIEW ARTICLE

New approaches in oral rotavirus vaccines Zenas Kuate Defo1 and Byong Lee1,2 Department of Microbiology and Immunology, McGill University, Montreal, Canada and 2School of Biotechnology, Jiangnan University, Wuxi, China

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Abstract

Keywords

Rotavirus is the leading cause of severe dehydrating diarrhea worldwide, and affects primarily developing nations, in large part because of the inaccessibility of vaccines and high rates of mortality present therein. At present, there exist two oral rotaviral vaccines, Rotarixä and RotaTeqä. These vaccines are generally effective in their actions: however, associated costs often stymie their effectiveness, and they continue to be associated with a slight risk of intussusception. While different programs are being implemented worldwide to enhance vaccine distribution and monitor vaccine administration for possible intussusception in light of recent WHO recommendation, another major problem persists: that of the reduced efficacy of the existing rotaviral vaccines in developing countries over time. The development of new oral rotavirus vaccine classes – live-attenuated vaccines, virus-like particles, lactic acid bacteria-containing vaccines, combination therapy with immunoglobulins, and biodegradable polymer-encapsulated vaccines – could potentially circumvent these problems.

Biodegradable polymers, combination therapy, lactic acid bacteria, rotaviral dehydrating diarrhea, virus-like particle

Introduction Rotavirus is the leading cause of severe dehydrating diarrhea worldwide, and it affects primarily young children under the age of five in developing nations in Asia and Africa. In fact, over 85% of these deaths have been found to occur in lowincome countries in Africa and Asia (Parashar et al., 2006). Strides have been made to eliminate this virus, as rotavirus vaccines have been introduced into national immunization programs in Sudan, Ghana, Rwanda, Malawi, Tanzania, parts of Zambia, Burundi, Burkina Faso, and The Gambia, with a further 14 African countries being deemed eligible for support, as per the Global Alliance for Vaccines and Immunizations (GAVI, 2014). In addition, India has developed its own rotavirus vaccine product and expects the product to be available at $1 a dose (Madhi & Parashar, 2014). In spite of this, the World Health Organization (WHO) estimates that rotavirus is responsible for 453 000 deaths annually (Tate et al., 2012). Because of the continued prevalence of rotaviral dehydrating diarrhea worldwide, it is imperative that additional, efficient rotavirus vaccines be produced. But why an oral rotavirus vaccine? Traditionally, vaccines are administered intravenously or by injection. Such vaccines – known as parenteral vaccines – are prepared using one of three methods. The first involves making a vaccine preparation from a dead micro-organism,

Address for correspondence: Dr. Byong Lee, Department of Microbiology and Immunology, Duff Medical Building, 3775 University St., Room 511, Montreal, QC H3A 2B4. Tel: (514) 3982835. E-mail: [email protected]

History Received 28 May 2014 Revised 24 August 2014 Accepted 3 September 2014 Published online 30 September 2014

after which the vaccine would be injected into the target individual’s bloodstream. Killing the micro-organism is useful, especially when dealing with pathogens that were highly virulent in their live state. An example of such a vaccine was Jonas Salk’s polio vaccine (Meldrum, 1998). Another common mode of vaccine preparation involved the use of live, attenuated micro-organisms. Such microorganisms have lost their virulence but retained the ability to stimulate the immune system. This technique was utilized when making the highly effective yellow fever vaccine, which consists of an attenuated strain of the yellow fever virus, 17D (Roukens et al., 2008). In yet another traditional vaccination technique, instead of taking the complete micro-organism, researchers have taken a portion of the micro-organism and used that in vaccine preparations. Such can be seen in the current day pneumococcal vaccine, which uses the Streptococcus pneumoniae bacterial polysaccharide as an immune system activator (Prymula et al., 2009). The aforementioned methods each have their perks and drawbacks. In general, vaccines that use live bacterial or viral products are extremely effective when they work, but carry a greater risk of causing disease. This is most threatening to individuals whose immune systems are impaired, such as individuals with leukemia (Ridgway & Wolff, 1993). Thus, children with leukemia are advised not to take the oral polio vaccine because they are at greater risk of developing the disease. Vaccines which do not include a live virus or bacteria tend to be safer, but their protection may not be as great. Yet another disadvantage of traditional vaccines is the fact that they are all limited by their dependence on biological

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products, which often must be kept cold, may have a limited life-span, and can be difficult to produce. Traditional vaccines, for the most part, act on the exterior of cells – however, in order to generate the best possible cellmediated response against a particular pathogen, it is often desirable to affect the target cells intracellularly. It was observed that antibody production could be induced following inoculation with a plasmid expressing human growth hormone (Tang et al., 1992). Thus, researchers turned to DNA, hoping that the intracellular nature of the product would produce greater immune responses and DNA vaccines were the result. The principle behind DNA vaccines is the following: microscopic portions of a virus’s DNA, encoding a viral antigen, are incorporated into a plasmid that is then injected into the patient. The patient’s own cells then adopt that DNA, and following transcription and translation produce the pathogenic viral proteins. These proteins are recognized as foreign, and as such, trigger an immune response. Such a technique has multiple advantages: high purity, the capacity to include multiple antigens, cost-effectiveness and suitability for use in the presence of pre-existing maternal immunity (Lavelle & O’Hagan, 2006). In addition, DNA vaccines, contrary to traditional viral vaccines, do not require live viral components, thus allowing for the circumvention of issues relating to vector immunity that could have deleterious effects on vaccine safety and immunogenicity. Another problem with traditional parenteral vaccines is the fact that they are not effective at increasing mucosal T-cell proliferation (Davenport et al., 2008). These vaccines lack the ability to produce an adequate immune response at the gut mucosal surface from the bloodstream (Holmgren & Czerkinsky, 2005). Initiating proper immune responses on gastrointestinal (GI) mucosal surfaces requires local delivery of the vaccine, which can be achieved via oral vaccination. The oral route has the advantages of avoiding the pain and discomfort encountered during parenteral and DNA vaccine injection and eliminating problems relating to inadequately sterilized needles and needle reuse. Such vaccines are also cheaper to administer, as there is no need for trained personnel, and to produce. However, the presence of proteolytic enzymes in the GI tract coupled with the low efficiency of transport across the epithelium makes for poor vaccine uptake in the intestine (Lavelle & O’Hagan, 2006). Thus, adjuvants, as well as other delivery systems, are required to enhance the immune response. At present, two oral rotavirus vaccines exist, RotarixÔ and RotaTeqÔ. While both have been deemed effective at clearing rotavirus infection (Ruiz-Palacios et al., 2006; Vesikari et al., 2006a), the need still exists for additional rotavirus vaccines. The next few pages of this comprehensive review will be organized as follows. First, aspects of rotavirus classification, pathogenesis, and distribution will be presented. Next, attention will be directed to current rotavirus vaccines, why new rotavirus vaccines are needed, and possible alternatives to present-day vaccines. Finally, future implications of these novel approaches in rotaviral vaccine development will be discussed.

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Rotavirus classification and pathogenesis Rotavirus is 70 nm in diameter, and its particles, as its name implies, resemble wheels (Latin, rota ¼ wheel). Classified as a genus within the family Reoviridae, they are non-enveloped and their capsids display icosahedral symmetry (Clark & McKendrick, 2004). The core or inner capsid, which is composed of viral capsid protein 2 (VP2) and contains the double-stranded RNA that is the viral genome, is surrounded by an inner (VP6) and outer capsid (VP7 and VP4). The genome consists of 11 segments which encode six viral capsid proteins (VP 1–4, 6, 7) and six non-structural proteins (NS 1–6). The proteins VP4 and VP7 constitute most of the outer capsid – VP4 is involved in cell attachment, while VP7 gives the virus its smooth surface (Clark & McKendrick, 2004; Gray et al., 2008). There exist eight rotavirus serogroups (A–H) (Clark & McKendrick, 2004; Matthijnssens et al., 2012). These groups are based on the antigenic properties of the inner capsid protein VP6, the most immunogenic protein in rotavirus infection (Parashar et al., 1998). In fact, sequence data for the aforementioned capsid protein from rotavirus serogroups A–D and F–H demonstrated that each serogroup shared at most 53% of their VP6 open reading frame amino acids – that is, rotavirus serogroups A–D and F–H were justified, in their categorization as serogroups, precisely because they respected the 53% amino acid cutoff value used to differentiate distinct rotavirus species (Matthijnssens et al., 2012). Among the eight, four serogroups – A, B, C, and H – have been found to be human pathogens, with group A rotavirus being responsible for the majority of human diseases (Matthijnssens et al., 2012; Parashar et al., 1998). Viruses within each serogroup can be further classified into serotypes on the basis of their outer capsid antigens. VP7 is a glycoprotein which defines the G-type antigens, and VP4 is a protease-cleaved protein which defines the P-type antigens: to date, 27 G- and 35 P-genotypes have been described in at least 73 G/P combinations (Abe et al., 2011; Matthijnssens et al., 2011; Matthijnssens & Van Ranst, 2012). VP4 is encoded by gene segment four, VP6 by gene segment six, and VP7 by gene segment seven, eight or nine, depending on the strain (Parashar et al., 1998). Rotavirus has been found to cause diarrhea by the following mechanism. It first invades the epithelium on the side and tips of the villi in the duodenum and upper jejunum, whereupon they then migrate into the cytoplasm of the enterocyte epithelial cells. This results in the destruction of the cells that synthesize disaccharidases. Thus, lactose and other disaccharides remain in the lumen of the bowel. Only monosaccharides are absorbed by the healthy bowel, resulting in osmotic drain, as body fluid is attracted into the bowel lumen. The diarrhea that ensues occurs mainly as a result of the luminal accumulation of lactose. Absorption of xylose is also impaired during rotavirus infection: as xylose and glucose are absorbed by the same pathway, glucose in excess can make the diarrhea worse. Furthermore, bacteria in the colon break down glucose and lactose into short-chain acids, thus greatly increasing the osmolarity of the contents in the colon. Therefore, colonic bacteria can further exacerbate the diarrhea (Flewett & Woode, 1978; Gray et al., 2008).

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More specifically, rotaviral infection of enterocytes is thought to be due to production of the viral enterotoxin NSP4, which is associated with an early secretory phase, resulting in ion secretion and massive fluid loss. This is followed by a milder and more prolonged late secretory phase, where cell destruction, transient carbohydrate intolerance, and nutrient malabsorption occur (De Marco et al., 2009).

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Rotaviral dehydrating diarrhea worldwide Rotaviral dehydrating diarrhea is a major global disease. In a study that examined the burden of severe rotavirus dehydrating diarrhea in Indonesia, it was found that of the 2240 children hospitalized for diarrhea, 1345 (60%) were rotaviruspositive. Moreover, dehydration and vomiting were more common in these patients than in their rotavirus-negative counterparts (Soenarto et al., 2009). Another study showcased the harsh reality of rotaviral dehydrating diarrhea in Africa – of the 381 deaths that occurred in children due to acute diarrhea in Ghana’s Kassena-Nankana District from 1998 to 2004, 131 (34%) were estimated to have been caused by rotavirus infection. The same study also established the need for rotavirus vaccine administration to all children, irrespective of their age. A 90% efficacious three-dose rotavirus vaccine was found to prevent 70% of deaths due to rotavirus infection if administered without age restrictions, while preventing 53% of deaths if initiated among children less than 12 weeks of age, and 52% of deaths if the course also was completed by 32 weeks of age (Arvay et al., 2009). While rotaviral dehydrating diarrhea is of primary concern in impoverished nations, it has also been found to affect developed countries. A study conducted in the US determined that rotavirus infection caused approximately 60 000 hospitalizations and 37 deaths annually. The bulk of these deaths occurred in minority children (Fischer et al., 2007). In sum, one can see that rotaviral dehydrating diarrhea is a worldwide phenomenon that hits hardest in areas where means are few. Because vaccine administration has been found to prevent a great number of deaths attributed to rotaviral dehydrating diarrhea, it is vital that effective and low-cost vaccines be produced.

Current rotavirus vaccines Currently, there are two vaccines used against rotavirus – RotarixÔ and RotaTeqÔ. Initially thought to be ineffective in developing countries, vaccine trials, conducted in South Africa and Malawi, have come to demonstrate that a live, oral rotavirus vaccine in fact significantly reduces episodes of severe rotaviral dehydrating diarrhea in African children (Madhi et al., 2010). Both are live, orally administered vaccines that mimic the protection offered by a rotavirus infection. While they have been found to have similar efficacy, immunogenicity, and safety, they do have different characteristics and modes of administration, which will be examined in the following few paragraphs. Rotarix Rotarix Ô is a human monovalent vaccine. It is composed of the single live rotavirus strain G1P[8] RIX4414, and was

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initially licensed in Mexico by GlaxoSmithKline (GSK) in July 2004. At present, RotarixÔ is licensed in over 100 countries worldwide (Ward & Bernstein, 2009). The final product also contains a calcium carbonate buffer (Bernstein, 2006), which protects the strain from the gastric acid in the stomach. The current RotarixÔ is a liquid formulation (Anh et al., 2011; Vesikari et al., 2011). It is administered in two doses. It is recommended the doses be administered to infants between the ages of 6 and 24 weeks, with the second dose occurring at least 4 weeks after the first (O’Ryan & Linhares, 2009; Zaman et al., 2009). The vaccine RotarixÔ replicates well in the gut, and provides protection similar to that obtained via natural infection. Vaccination with RotarixÔ provides the host with homotypic antibodies, and subsequent infection by other rotaviruses leads to a broadening of the immune response, which provides cross-protection against most other serotypes (Kirkwood, 2010). Thus, effectiveness of RotarixÔ relies on the concept of ‘‘heterotypic immunity’’: cross-protection is conferred by natural rotavirus infection of one VP7/VP4 combination against subsequent symptomatic reinfection caused by a different VP7/VP4 combination, with both viruses sharing only one of the capsid antigens (O’Ryan, 2007). A question remains, however, as to whether Rotarix will be able to provide protection against completely heterotypic strains whose genotype combinations are foreign to monovalent G1P[8] rotavirus strains (Kirkwood, 2010). RotaTeq The other rotavirus vaccine currently in use is RotaTeqÔ. It (licensed by Merck in 2006) is a pentavalent rotavirus vaccine that consists of five human-bovine (WC3) reassortant rotaviruses suspended in a buffered liquid which protects the strains from gastric acid (Heaton & Ciarlet, 2007; Vesikari et al., 2006a). For each reassortant, the WC3 core associates with a human rotavirus surface protein – G1, G2, G3, G4, or P1A[8]. In contrast to RotarixÔ, RotaTeqÔ is administered orally in three doses. The first dose is administered at 6–12 weeks of age, the second dose follows at a 4–10 week interval, and the last dose is administered by the time the infant is 32 weeks of age (Heaton & Ciarlet, 2007). RotateqÔ relies on the concept that each reassortant will generate a particular class of neutralizing antibodies against specific rotavirus types contained within the vaccine. Unlike RotarixÔ, the background strain used for RotateqÔ – the bovine WC3 reassortant rotavirus – does not grow well in humans, and is not broadly protective. This implies that if the most common disease-causing rotaviruses are absent from the vaccine composition, then the vaccine composition would potentially need to be reformulated (Kirkwood, 2010). Problems with the present-day rotavirus vaccines As mentioned, present-day rotavirus vaccines have been quite effective in their ability to protect children from infection. A minor default is their association with intussusception in infants with intestinal malformations or genetic traits which predispose for intussuception (Lepage & Vergison, 2007). A recent study, conducted in the USA, has

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shown that the rate of intussusception significantly increased among infants who had been vaccinated with the monovalent rotavirus vaccine, from 0.72 (as per historical data) to 5.3 cases per 100 000 infants vaccinated (Weintraub et al., 2014). Similar findings have been reported in Mexico and in Brazil for the monovalent rotavirus vaccine (1 of every 51 000 to 68 000 vaccinated infants were at short-term risk of intussusception) (Patel et al., 2011; Vela´squez et al., 2012). The pentavalent vaccine has also been purported to present an increased risk of intussusception in Australia, where the vaccine attributable risk for intussusception was estimated to be 7.0 cases per 100 000 infants vaccinated (Carlin et al., 2013), and in the USA (3.75 cases per 100 000 vaccinated infants; 1.5 excess cases of intussusception per 100 000 infants) (Haber et al., 2013; Yih et al., 2014). It should be noted that these studies acknowledge possible limitations in their findings stemming from differences in the background rates of intussuception, differences in the methodology applied, and deficient sample sizes (Bines et al., 2009; Justice et al., 2005; Mullooly et al., 1999; Weintraub et al., 2014; Yih et al., 2014), and as such, further research is needed into the relationship between intussusception and the current oral rotavirus vaccines. Moreover, the aforementioned studies mention that the benefits of rotavirus vaccination outweigh the risk of intussusception: nevertheless, the potential for intussuception is being monitored by the creation of surveillance systems in developing countries, such as those found in Africa (Steele et al., 2012), especially in the wake of recommendations by the WHO that the age window for vaccine administration be relaxed (Tate et al., 2010). Another potential, albeit minor (because of extensive clinical trials demonstrating the effectiveness of the two present-day rotavirus vaccines (Ruiz-Palacios et al., 2006; Vesikari et al., 2006a)) problem stems from trends in rotavirus vaccine history. Prior to both present-day vaccines, there existed another licensed live, orally-deliverable vaccine, RotaShieldÔ. In 1999, following the discovery of its association with intussusception (Murphy et al., 2003), there existed a void of 5 years until the next rotavirus vaccine, RotarixÔ, was licensed. In order to minimize the risk of ever having a similar gap in time during which no rotavirus vaccine is present, it is prudent other rotavirus vaccines be developed; moreover, it would be wise to produce non-live vaccines, for if it were found the current vaccines triggered intussusception over time – unlikely, but possible – then all live, oral rotavirus vaccines would be deemed unsafe (Ward et al., 2008). The current vaccines also present problems in terms of supply and high costs. Increased vaccine production by GSK and Merck, commitment to rotavirus vaccine purchasing in developing countries by GAVI, and competition and developing world manufacturers creating new products – such as the alternative live oral rotavirus vaccines 116E (India) and RV3 (Australia, Indonesia) mentioned in the following section – are helping to alleviate these problems (Glass et al., 2005). A major problem, however, is the reduced efficacy of these oral vaccines in developing countries. Recent studies, while demonstrating that rotavirus vaccines reduced severe episodes of rotavirus-induced gastroenteritis in African neonates, also found that the overall efficacy of the vaccine in preventing these episodes was lower than that observed in European and

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Latin American studies (Madhi et al., 2010, 2012). Similarly, it was found in a randomized, double-blind, placebocontrolled trial conducted in Ghana, Kenya, and urban Mali that efficacy of the pentavalent rotavirus vaccine beyond the first year of life declined from the first to the second year – findings which differed with European and American studies, where declines in efficacy from the first to the second year of life and beyond were minute (Armah et al., 2010).

Alternative live oral rotavirus vaccines RV3 Human rotavirus vaccine RV3 is a G3P[6] strain that was obtained from infant feces in Australia. It shares an immunodominant neutralizing epitope on VP7 with G1 strains (Barnes et al., 1997). Previous studies have shown that babies infected with this RV3 vaccine produced persisting neutralizing antibodies to G1, G3 and G4 strains at 3 months of age (Bishop et al., 1990). Furthermore, babies receiving the RV3 vaccine were protected against clinical diseases due to G2 rotavirus strains during the first 3 years of life (Programme for Control of Diarrhoeal Diseases [PCDD], 1988). The vaccine has undergone two trials thus far. Phase I trials indicated the virus was a naturally attenuated rotavirus strain, that the vaccine stimulated IgA responses, and that the candidate vaccine could potentially stimulate immune responses to G3 and G1 viruses simultaneously (Barnes et al., 1997). Phase II trials have shown the protection conferred by RV3 vaccination is heterotypic (Barnes et al., 2002). The latest data for the RV3-BB rotavirus vaccine indicate safety in adults, children and infants: most infants receiving the vaccine only required a single dose to elicit an appropriate response, supporting the continued progression of the vaccine candidate to phase II immunogenicity and efficacy trials (Danchin et al., 2013). 116E The prototype strain 116E belongs to serotype G9 and genotype P[11] (Das et al., 1994). It is believed to be a human-bovine reassortant strain (Gentsch et al., 1993). Similar to Rotarix, it is derived from a common and virulent human strain attenuated by serial passages in cell culture. The findings of a recent safety and immunogenicity trial indicate that the responses observed following a single dose of the 116E vaccine are at least as great as those found after the first dose of Rotarix (Bhandari et al., 2006). Human rotavirus vaccines 116E and RV3 were both found to contain amino acid residues that could influence the attenuation and efficacy of each vaccine (Rippinger et al., 2010). The UK bovine/human reassortant vaccine The UK/bovine reassortant vaccine contains single VP7 gene substitutions from G1, G2, G3 or G4 human rotaviruses on a 10-gene UK background. A safety and immunogenicity trial conducted in the USA showed that two doses of the vaccine were necessary to infect 100% of the patients and provide them with satisfactory levels of attenuation, safety, infectivity, and immunogenicity (Clements-Mann et al., 1999). A later

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trial in Finland confirmed the findings of the USA trial (Vesikari et al., 2006b). However, the final formulation of the vaccine has yet to be tested. A much larger study is also in the works to address the safety of the vaccine with regard to intussusception.

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Virus-like particles Virus-like particles (VLPs) are useful tools for vaccine development. Their oral administration has elicited immune responses in humans against other enteric viruses, such as the Norwalk virus (Tacket et al., 2003). They contain viral spikes and other surface components in a repetitive array, which efficiently stimulates T-cell and B-cell responses (Chackerian, 2007). Rotaviral VLPs are produced by co-infecting Spodoptera frugiperda 9 (Sf9) cells with different combinations of baculovirus recombinant plasmids. Various combinations of these recombinants – BacRf2A, pVL941/SA11-4, pAc461/ SA11-6, and pVL941/SA11-9 or pVL1392/HRV8697-9 – produced different particles: VP2/6, VP2/4/6, VP2/6/7, and VP2/4/6/7 (Crawford et al., 1994). These particles were found to be extremely stable: EM analysis of the purified particles stored at 4  C for at least a year revealed the particles remained intact (Crawford et al., 1994). Research has shown that rabbits vaccinated with VP2/4/6/ or VP2/6/7 VLPs were either totally or partially protected from homotypic G3 rotavirus challenge. G1 VP2/6/7 VLPs were able to generate the same neutralizing antibodies to both G1 and G3 viruses as VP2/4/6/7, suggesting it might be possible to reduce the number of G-types incorporated into a subunit vaccine (Conner et al., 1996). It has even been shown that VP2/6 can provide partial protection against rotavirus infection when coupled with an adjuvant (Zhou et al., 2011). By reducing the number of vaccine components, one may then reduce vaccine production costs. However, it has also been shown that the protective efficacy induced by VLPs was significantly less than that induced by the attenuated rotavirus. This suggests that additional viral components are necessary to elicit a more robust protective response against rotavirus infection (Blutt et al., 2006).

Lactic acid bacteria as vehicles for efficient oral vaccine delivery Over the past few years, much has been made of the use of lactic acid bacteria (LAB) as possible live vaccine delivery vectors because of their ability to stimulate the immune system. Lactic acid bacteria are used for the preservation and fermentation of industrial foods, and have beneficial effects on human health – hence the designation ‘‘Generally Regarded As Safe’’ (GRAS). Experimental and clinical evidence has been elucidated, demonstrating enhanced antimicrobial immune protection when probiotic LAB are present (Cross, 2002). LAB, and in particular lactobacilli, have also been found to (1) influence the immune response in a variety of ways, depending on the strain used, and (2) make great cloning vectors for the delivery of antigens (Seegers, 2002). There are two main ways in which lactobacilli allow for efficient delivery of rotavirus vaccines. They may either

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(1) serve as a vector for vaccine transport (often transporting the vaccine as a recombinant vaccine), or they may (2) stimulate the target organ to be more receptive to the vaccine. Examples of the former include the recombinant porcine rotavirus VP4 vaccine expressed in L. casei (Qiao et al., 2009), and oral immunization with live L. lactis expressing the rotavirus VP8 subunit (Marelli et al., 2011). Examples of the latter are explained in the following paragraphs. In a recent study, gnotobiotic pigs were vaccinated with an attenuated human rotavirus vaccine, and the resultant immune response was characterized and quantified (Zhang et al., 2008). The IFN-g producing CD8+ T cells frequency in gnotobiotic pigs vaccinated and fed Lactobacillus acidophilus were found to be significantly higher in the ileum, spleen and blood than in their L. acidophilus-deficient counterparts (Figure 1a). Thus, feeding of L. acidophilus significantly increased the intestinal and systemic human rotavirus-specific IFN-g producing CD8+ T cell responses induced by the attenuated human rotavirus. Both intestinal human rotavirus-specific and total intestinal IgA-secreting cell responses (SCR) were also enhanced in L. acidophilus-fed vaccinated pigs, with the rotavirusspecific response being significantly enhanced vis-a`-vis their L. acidophilus-deficient counterparts (Figure 1b and c). Similarly, IgG-SCR were also increased in L. acidophilusfed vaccinated pigs (Figure 1b and c). Therefore, pigs fed L. acidophilus had enhanced IgA and IgG intestinal responses. A more recent study, demonstrating how different doses of L. acidophilus exert differential immunomodulatory effects on T cell immune responses following rotavirus vaccination in gnotobiotic pigs (Wen et al., 2012), serves as yet another example of how LAB may stimulate mucosal immunity, rendering the intestine more amenable to the vaccine.

Combination therapy with immunoglobulins and probiotics as a prophylaxis Little has been written in the past about the prospects of combination therapy as a means of generating a rotaviral vaccine. However, recent findings suggest this might be another possible route one may take to produce the desired rotavirus vaccine. Combination therapy, using immunoglobulins and probiotics as a prophylaxis, operates based on the following two principles. The first is that if we provide an individual at risk of a particular infection with antibodies from an external source, then these antibodies may be able to provide protection against the infection via passive immunity. The passive transfer of maternal secretory IgA (sIgA) through breast milk has been shown to have important implications in the neonatal period for protection against a wide variety of pathogens, including rotavirus. This generalization, however, is not entirely applicable to neonates in developing countries: in South Africa, for instance, it was found that infants who did not receive breast milk, for 1 h before and after rotavirus vaccination, had no significant changes in their immune response to the vaccine, as compared to their breast milkreceiving counterparts (Groome et al., 2014). Thus, transfer of passive immunity against rotavirus through orally delivered immunoglobulins is a viable prophylactic strategy for

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Figure 1. Lactobacillus acidophilus upregulates anti-rotaviral immune responses by increasing T-cell IFN-g frequency and rotavirus-specific intestinal antibody responses. (a) HRV-specific IFN-g producing T cell responses in the Gn pigs vaccinated with attenuated human rotavirus (AttHRV) with or without L. acidophilus (LA). Mononuclear cells from the ileum, spleen and peripheral blood of pigs were extracted and assayed on PID 28. Frequencies of IFN-g + CD8+ T cells among CD3+ MNC were determined by an intracellular staining and flow cytometry assay after the MNC were stimulated with purified AttHRV antigen for 17 h. Frequencies from the mock-stimulated MNC were subtracted from the frequencies of the AttHRV-stimulated MNC. Data represent the adjusted mean frequency (n ¼ 4 for LA + AttHRV+; n ¼ 7 for LA  AttHRV+) of HRV-specific IFN-g producing T cells. Bars with different letters (A and B) on top differ significantly for the same tissue (Kruskal–Wallis test, p50.05). Reprinted with permission from Zhang et al. (2008). Probiotic Lactobacillus acidophilus enhances the immunogenicity of an oral rotavirus vaccine in gnotobiotic pigs. Vaccine 26:3655–61. (b, c): HRV-specific ASC and total Ig SC responses in Gn pigs vaccinated with AttHRV with or without LA. Mononuclear cells from the ileum, spleen and peripheral blood of pigs were extracted and assayed on PID 28. Enzyme-linked-immunospot assays for determining HRV-specific ASC and total IgSC numbers were performed on the day of MNC extraction. Data represent the mean numbers of HRV-specific ASC (b) and total IgSC (c) per 5  105 MNC, respectively (n ¼ 4–7). Bars with different letters (A–C) on top differ significantly among groups for the same tissue and the same antibody isotype (Kruskal–Wallis test, p50.05). Reprinted with permission from Zhang et al. (2008). Probiotic Lactobacillus acidophilus enhances the immunogenicity of an oral rotavirus vaccine in gnotobiotic pigs. Vaccine 26:3655–61.

developed countries, with further testing needed in developing countries. The second principle is that probiotics, such as the LAB seen in the previous section, by virtue of their being found in large numbers in our intestines, can be used as safe delivery vehicles of the antibodies to the target organ, where they will stimulate the mucosal immune system to release secretory IgA (Kaila et al., 1995). Recently, a research team in Sweden has been looking at the combination of llama antibodies and Lactobacillus rhamnosus GG, and its ability to confer protective immunity against rotavirus infection. Genetically engineered antibody fragments (GEAFs) have been found to make possible sources of therapy for rotavirus infection. Variable domain of llama heavy-chain antibodies (VHH) are one such example. In contrast to other GEAFs, these antibodies are smaller, much more heat and acid resistant, exhibit high epitope-specific affinity, and while in the GI tract maintain intact spatial structure (Harmsen & De Haard, 2007). VHH antibody fragments also have longer projecting CDR loops than conventional immunoglobulins, which allow for more efficient targeting of cryptic immune-evasive sites (Stijlemans et al., 2004). It was shown that VHH secreted by yeast (van der Vaart et al., 2006) and lactobacilli (Pant et al., 2006) were effective in neutralizing rotavirus. Not only were lactobacilli-secreted VHH effective in neutralizing rotavirus, they also effectively reduced viral load, normalized pathological features and alleviated diarrhea when anchored to lactobacilli (Figure 2) (Pant et al., 2006). These same lactobodies have also been found to be effective at reducing morbidity in rotavirusinduced diarrhea in mice in vivo (van der Vaart et al., 2006). Moreover, it was found that combinations of llama antibodies could be used to impart more robust protection against rotaviral infection than single antibody fragments (Pant et al., 2011). It still remains to be seen how effective VHH is in Asia and Africa.

Another GEAF is the bovine antibody. Pregnant cows, when immunized, produce a high concentration of epitopespecific antibodies in their colostrum, making them attractive choices for high-scale antibody production. The antibodies produced are called hyper-immune bovine colostrums (HBCs), and have been successfully used for the treatment of enterotoxigenic and enterophathogenic Escherichia coli infections, as well as therapeutically against rotavirus (Casswall et al., 2000). Recently, it was shown that the combination of bovine antibodies and lactobacilli effectively provided prophylaxis against rotaviral dehydrating diarrhea (Pant et al., 2007). HBCs have excellent stability profiles and, once lyophilized, do not require special storage conditions. Despite being a highly effective treatment method, production costs of HBCs are quite high. Thus, alternative, less costly sources of antibodies are needed. As mentioned previously, lactobacilli stimulate numerous strong anti-rotaviral immunological responses. Studies are presently being conducted to see whether or not additional lactobacilli can be transformed into other genetically engineered probiotics that produce antibody fragments. The results look promising (Ko˜ll et al., 2010; Martı´n et al., 2011).

PLA- and PLGA-encapsulated rotavirus particles The possibility of encapsulated rotavirus particles serving as rotaviral vaccines has been studied extensively in the past (Chen et al., 1998; Sturesson et al., 1999). Controlled delivery of viral particles using biodegradable polymers such as polylactide (PLA) and poly(lactic-co-glycolic acid) (PLGA) provide another plausible alternative to present-day vaccines, as the delivery system is easy to administer and cost effective (O’Hagan et al., 1998). PLA and PLGA can deliver their encapsulated particles to desired locations at pre-determined rates, as well as for specific amounts of time, in order to produce an optimal immune response (O’Hagan et al., 1991). These polymers also degrade in the body to non-toxic,

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Figure 2. The variable domain of llama heavy-chain antibodies provides protection against rotavirus pathogenicity. (a) In vitro rotavirus neutralization assay showing the reduction in infection rate achieved by the variable domain of llama heavy-chain (VHH1) produced by lactobacilli in its secreted form. Reprinted with permission from Pant et al. (2006). Lactobacilli expressing variable domain of llama heavy-chain antibody fragments (lactobodies) confer protection against rotavirus-induced diarrhea. J Infect Dis 154:1580–88. (b) Evaluation of the efficacy of diarrhea reduction of different doses of variable domain of llama heavy-chain (VHH1)-anchored lactobacilli. On day 2, 1  108 cfu (n ¼ 7) and 1  109 cfu (n ¼ 8) of VHH1anchored lactobacilli caused a significant reduction in diarrhea prevalence, compared with that in the untreated group (n ¼ 10) (p50.0001 and p ¼ 0.0024, respectively). What’s more, 1  109 cfu/dose non-transformed Lactobacillus paracasei (n ¼ 10) and 1  107 cfu/dose of VHH1-anchored lactobacilli (n ¼ 7) did not show any significant reduction relative to the untreated group. Pooled results of three experiments (n ¼ 27 VHH1-anchored lactobacilli, n ¼ 17 wild-type, n ¼ 17 irrelevant VHH-anchored lactobacilli directed against the Streptococcus mutans SA I/II adhesin, n ¼ 10 VHH1secreted lactobacilli, and n ¼ 30 untreated). VHH1-anchored lactobacilli significantly reduced diarrhea prevalence on days 2 and 3 relative to untreated lactobacilli (p50.002 and p50.0001, respectively, Fisher’s exact test) and on day 2 relative to irrelevant VHH-anchored lactobacilli (p50.03, Fisher’s exact test). Reprinted with permission from Pant et al. (2006). Lactobacilli expressing variable domain of llama heavy-chain antibody fragments (lactobodies) confer protection against rotavirus-induced diarrhea. J Infect Dis 154:1580–88. (c) Load of vp7 RNA in small intestinal tissue samples as determined by real-time polymerase chain reaction (PCR). Mice were challenged with 20 diarrhea doses on day 0; on day 4 after infection, the virus load in the variable domain of llama heavy-chain (VHH1)-anchored lactobacilli treatment group was significantly reduced, relative to the group treated with irrelevant lactobacilli. *p50.05, Mann–Whitney U test. The reverse-transcription PCR analyses were repeated twice for all the samples, and similar results were obtained. Reprinted with permission from Pant et al. (2006). Lactobacilli expressing variable domain of llama heavy-chain antibody fragments (lactobodies) confer protection against rotavirus-induced diarrhea. J Infect Dis 154:1580–88.

low-molecular-weight products that are easily eliminated from the body via the Krebs cycle (Glass et al., 1996). Finally, the polymers have variable hydrophobicity, which allows for better interaction with antigen-presenting cells, and adjuvant properties for improved immunogenicity of the entrapped antigen (Glass et al., 2006; Shaobing et al., 2004). Briefly, PLA- and PLGA-encapsulated rotavirus particles are formed by a water-in-oil-in-water multiple emulsion technique (Raghuvanshi et al., 2001). In the primary emulsion, rotaviral antigen is combined with a polymer solution. Following treatment with polyvinyl alcohol and sucrose, the secondary emulsion is prepared, this time by sonicating the primary aqueous phase with an external aqueous phase. Overnight stirring and washing produces the finalized particles (Nayak et al., 2009). A recent study has shown PLA-encapsulated rotavirus particles elicit significant antibody responses, which were sustained for longer periods of time than their free-rotavirus

counterparts (Nayak et al., 2009). Another recent study showed PLA-encapsulated rotavirus microparticles elicited a better response than PLGA-encapsulated rotavirus microparticles when administered through the oral route, and that the PLGA-encapsulated rotavirus microparticles elicited better antibody responses through the intranasal route (Nayak et al., 2011). While it was shown the performance of intranasal route-based immunization was significantly higher than the oral and intramuscular routes (Nayak et al., 2011), the fact that a significant improvement in immunogenicity was observed upon oral administration suggests that rotaviral particle entrapped in biodegradable polymers could possibly be another source of oral rotavirus vaccines.

Discussion and conclusions The present-day oral rotavirus vaccines, while effective, have been associated with intussusception

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Table 1. Alternative oral rotavirus vaccines and vaccine candidates. Candidates

Properties

RotarixÔ RotaTeqÔ

Single monovalent rotavirus strain G1P[8] RIX4414 Pentavalent vaccine of five human-bovine (WC3) reassortant rotavirus core associated with G1, G2, G3, G4, or P1A[8] G3P[6] rotavirus strain Rotavirus strain belonging to serotype G9 and genotype P[11] Single VP7 gene substitutions from G1, G2, G3, or G4 human rotaviruses on a 10-gene UK background Particles formed from expressed recombinant proteins. Contain VP2 and VP6 with or without VP4 and VP7 LAB (often lactobacilli) serving as an immunostimulatory vector for the rotavirus vaccine Llama or bovine antibodies Polylactide (PLA) and poly(lactic-co-glycolic acid) (PLGA)encapsulated rotavirus particles

RV3 116E UK reassortant Virus-like particles (VLPs) Lactic acid bacteria (LAB)

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Combination therapy Biodegradable polymer-encapsulated vaccines

Status Licensed Licensed Phase I and early Phase II Phase I and early Phase II Phase I and early Phase II Tested in mice, rabbits, and gnotobiotic pigs Tested in gnotobiotic pigs Tested in mice Tested in mice

Adapted with permission from Ward et al. (2008).

(Lepage & Vergison, 2007). Moreover, they prove to be too costly – a fact which has been claimed by some to be the most challenging and crucial factor for decision-makers regarding whether to introduce this vaccine into developing countries’ immunization schedules (Tu et al., 2011). Advances have been made to increase vaccine distribution worldwide. For developing countries, however, it appears the greatest problem presented by these vaccines is their reduced efficacy, visa`-vis developed countries. New vaccines are thus needed that will be effective in spite of possible host malnutrition, enteric coinfections, and waning immunity(Armah et al., 2010; Madhi et al., 2010), while reducing the risk of intussusception from slight to none. This review thus aimed to shed some light on the novel developments pertaining to oral rotavirus vaccines (summarized in Table 1). Three live-attenuated vaccines – RV3, 116E, and the UK bovine/human reassortant – are presently in phase II trials or beyond. Of all the possible oral vaccine alternatives listed in this review, the live-attenuated vaccines appear at present to stand the greatest chance of becoming commercial vaccines – no other vaccine variety was found in the literature to be beyond phase I trials. These vaccines, by virtue of them being comprised of the infectious virus in its entirety, and not a fragment of the virus, are most likely to provide complete protection against rotaviral dehydrating diarrhea, explaining why great emphasis is being placed on the development of these candidate vaccines. Live vaccines, in essence, elicit the most effective protective responses since they stimulate both the systemic and mucosal immunity (Kodama et al., 2005; Uren et al., 2005). While the development of new liveattenuated candidate vaccines is likely to drive down rotaviral vaccine prices (due to increased competition), vaccine storage requirements, production costs, and their limited life-spans will incur additional costs, which may or may not be counterbalanced by the decrease in costs associated with competition among vaccines. VLPs have the ability to act prophylactically against rotavirus infection. While oral vaccination with VLPs is possible, it appears they are more effective as an intranasal vaccine. VLPs do not pose the same threats as live-attenuated vaccines (notably that of mutating into a more virulent form): however, they also present the problem of not generating the same robust immune response as the live vaccine. VP6, by

virtue of it being the major immunogenic rotaviral protein, was present in most VLP concoctions – however, it was also commonly noted that other molecules were needed to complement the central immunogenic protein. This makes the VLP a less feasible option for the time being, as efforts must still be made to determine which combination of VLPs will produce the most robust response. Minimizing the number of VLPs in the complete formulation will allow for lower production costs. A positive feature of VLPs is that they remain intact for long periods of time at reasonable temperatures, which may make them an economically feasible option. Yet, work still must be done to determine how to obtain the optimal response from the smallest number of particles possible. The use of LAB as a delivery vehicle for vaccines occurs via the use of such bacteria as (1) transport vectors, and/or (2) immunostimulatory organisms. An advantage of LAB use is that they are organisms which are generally regarded as safe. Moreover, the fact that LAB have played a major role in the production of our food worldwide for a long time suggests that food industries across the globe are well experienced in the production of LAB cultures, and that their preparation on a large scale for incorporation into a rotaviral vaccine would thus be cost-effective. Another way in which LAB increases cost-effectiveness is by stimulating the mucosal immune system above and beyond solely viral antigen levels – in so doing, a smaller dose of viral antigen may be used to generate the same immune response. However, similar to VLPs, further studies with larger sample sizes, as well as human trials, are needed in order to determine the feasibility of this approach. Combination therapy utilizes both antibodies as a means of providing passive immunity and LAB as an immunostimulatory vaccine delivery system. The cost effectiveness of this approach is two-fold. First, the genetically engineered lactobacilli which produce these immunoglobulins have a long shelf life when they are lyophilized, simple logistics for distribution, and ease of administration. Second, the in situ production of VHH antibody fragments locally in the GI tract of the host circumvents the practical problem of the degradation of orally administered antibodies in the stomach while reducing the cost of antibody purification (Pant et al., 2006). It has been shown using a combination of immunoglobulins may have a synergistic effect (Pant et al., 2011);

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however, more studies are needed with larger sample sizes before reaching any definitive conclusions. Moreover, human trials are needed to determine the effectiveness of this method in humans. Rotavirus particles encapsulated with biodegradable polymers present another possible route for oral vaccine production. PLA and PLGA polymers possess numerous traits which make them favorable candidates for vaccine transport: controlled delivery of viral particles, delivery of viral particles to desired locations at pre-determined rates and specific amounts of time, degradation to non-toxic compounds, and adjuvant properties which increase vaccine efficiency. However, the conventional formulation of such polymers is hard to produce – this would therefore incur additional production costs. Furthermore, certain polymeric materials have been found to have cellular toxicity. Further studies are needed to understand the kinetics of viral particle release, as well as how to modify interactions of the viral particle with the polymeric carrier for optimum transfection efficiency without sacrificing biocompatibility (Xiang et al., 2010). In conclusion, it appears there are many possible routes by which oral rotavirus vaccines may be developed. The most feasible at present are the live-attenuated vaccine candidates. It has been suggested that the advent of these attenuated vaccine candidates will not offer significant improvement of protection over the present-day vaccines (with the exception of the UK bovine/human reassortant, which may contain additional reassortant viruses not present in RotaTeq) (Ward et al., 2008). That said, the presence of multiple vaccine manufacturers will inevitably drive down the cost of vaccines, making them more accessible to all children. Thus, multiple different approaches for oral rotavirus vaccine delivery are needed.

Declaration of interest The authors declare no conflicts of interests. The authors alone are responsible for the content and writing of this article.

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