New-Generation Treatment? Targeted Antiviral Therapy for Rotavirus

See “Anti-rotavirus protein reduces stool output in infants with diarrhea: a randomized placebo-controlled trial,” by Sarker SA, Jäkel M, Sultana S, et al, on page 740. See “Anti-rotavirus protein reduces stool output in infants with diarrhea: a randomized placebo-controlled trial,” by Sarker SA, Jäkel M, Sultana S, et al, on page 740. Rotavirus gastroenteritis was the leading cause of severe acute gastroenteritis in all parts of the world until the introduction of rotavirus vaccines. It remains the leading cause of gastroenteritis in developing countries, but in some industrialized nations, rotavirus is now second to norovirus infection.1Hemming M. Räsänen S. Huhti L. et al.Major reduction of rotavirus, but not norovirus, gastroenteritis in children seen in hospital after the introduction of RotaTeq vaccine into the National Immunization Programme in Finland.Eur J Pediatr. 2013; 172: 739-746Crossref PubMed Scopus (162) Google Scholar The introduction of live oral vaccines for rotavirus into national immunization programs has resulted in a dramatic reduction of rotavirus-associated gastroenteritis requiring hospitalizations in industrialized countries. A lesser reduction in rotaviral disease, which is still remarkable, has been described from middle-income countries.2Lopman B.A. Payne D.C. Tate J.E. et al.Post-licensure experience with rotavirus vaccination in high and middle income countries; 2006 to 2011.Curr Opin Virol. 2012; 2: 434-442Crossref PubMed Scopus (44) Google Scholar In lower income countries, the efficacy of oral rotavirus vaccines is low, ranging from 20% to 65%. Although the vaccines still have a large public health impact in the low-income or developing countries in Asia and Africa because of the high burden of disease, between one third and one half of all vaccinated children are not protected by currently available vaccines.3Babji S. Kang G. Rotavirus vaccination in developing countries.Curr Opin Virol. 2012; 2: 443-448Crossref PubMed Scopus (46) Google Scholar In such countries, availability of appropriate therapy continues to be a need for management of severe rotavirus gastroenteritis. In this issue, Sarker et al4Sarker S.A. Jäkel M. Sultana S. et al.Anti-rotavirus protein reduces stool output in infants with diarrhea: a randomized placebo-controlled trial.Gastroenterology. 2013; 145: 740-748Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar report the results of a randomized, double-blind, placebo-controlled trial of a biological molecule in the treatment of rotavirus diarrhea in Bangladesh. The main clinical manifestation of rotavirus infection is gastroenteritis, which may be mild, but can frequently be severe and life threatening, particularly in young children, who may dehydrate very quickly. Rotaviruses cause diarrhea in a multifactorial pathogenetic process that follows viral replication in the mature enterocytes of the small intestine.5Greenberg H.B. Estes M.K. Rotaviruses: from pathogenesis to vaccination.Gastroenterology. 2009; 136: 1939-1951Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar Diarrhea and malabsorption are believed to result from virus-mediated destruction of enterocytes leading to a reduction of epithelial surface area, virus-induced down-regulation of the expression of absorptive enzymes, paracellular leakage owing to functional changes in tight junctions, and the secretion of fluid and electrolytes through activation of the enteric nervous system and the effects of NSP4—the first described virus-encoded enterotoxin.5Greenberg H.B. Estes M.K. Rotaviruses: from pathogenesis to vaccination.Gastroenterology. 2009; 136: 1939-1951Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar Compared with bacteria, there are a limited range of antiviral agents available, and none are specifically targeted at enteric viruses. For rotavirus, the standard of care has been oral or intravenous rehydration. In the past 15 years, small trials have been reported with racecadotril, an enkephalinase inhibitor, and nitazoxanide, a drug reported to have a broad spectrum of activity against protozoa and several viruses (Table 1), some of which were sponsored by the manufacturer. All trials had relatively small numbers of children, not all were tested on children infected with rotavirus alone, and the studies evaluated different outcome measures; however, beneficial effect on ≥1 outcome measure was demonstrated.6Rossignol J.F. Abu-Zekry M. Hussein A. et al.Effect of nitazoxanide for treatment of severe rotavirus diarrhoea: randomised double-blind placebo-controlled trial.Lancet. 2006; 368: 124-129Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 7Teran C.G. Teran-Escalera C.N. Villarroel P. Nitazoxanide vs. probiotics for the treatment of acute rotavirus diarrhea in children: a randomized, single-blind, controlled trial in Bolivian children.Int J Infect Dis. 2009; 13: 518-523Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 8Cézard J.P. Duhamel J.F. Meyer M. et al.Efficacy and tolerability of racecadotril in acute diarrhea in children.Gastroenterology. 2001; 120: 799-805Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 9Salazar-Lindo E. Santisteban-Ponce J. Chea-Woo E. et al.Racecadotril in the treatment of acute watery diarrhea in children.N Engl J Med. 2000; 17: 463-467Crossref Scopus (224) Google Scholar, 10Lehert P. Chéron G. Calatayud G.A. et al.Racecadotril for childhood gastroenteritis: an individual patient data meta-analysis.Dig Liver Dis. 2011; 43: 707-713Abstract Full Text Full Text PDF PubMed Scopus (53) Google ScholarTable 1Efficacy of Newer Treatments (Excluding Probiotics) on Hospitalized Children With Rotavirus GastroenteritisLocation of studyNumber of rotavirus positive children/total numberAge/genderComparison groupsMain outcomeReferenceEgypt38/505 mos–7 y; male and femaleNitazoxanide vs placeboDuration of diarrhea (60% reduction)6Rossignol J.F. Abu-Zekry M. Hussein A. et al.Effect of nitazoxanide for treatment of severe rotavirus diarrhoea: randomised double-blind placebo-controlled trial.Lancet. 2006; 368: 124-129Abstract Full Text Full Text PDF PubMed Scopus (157) Google ScholarBolivia751–24 mos; male and femaleNitazoxanide vs placeboDuration of diarrhea (24% reduction)7Teran C.G. Teran-Escalera C.N. Villarroel P. Nitazoxanide vs. probiotics for the treatment of acute rotavirus diarrhea in children: a randomized, single-blind, controlled trial in Bolivian children.Int J Infect Dis. 2009; 13: 518-523Abstract Full Text Full Text PDF PubMed Scopus (95) Google ScholarFrance67/1723 mos–4 y; male and femaleRacecadotril vs placeboStool output (40% reduction)8Cézard J.P. Duhamel J.F. Meyer M. et al.Efficacy and tolerability of racecadotril in acute diarrhea in children.Gastroenterology. 2001; 120: 799-805Abstract Full Text Full Text PDF PubMed Scopus (113) Google ScholarPeru73/1353–35 mos; maleRacecadotril vs placeboStool output (46% reduction)9Salazar-Lindo E. Santisteban-Ponce J. Chea-Woo E. et al.Racecadotril in the treatment of acute watery diarrhea in children.N Engl J Med. 2000; 17: 463-467Crossref Scopus (224) Google ScholarIndia41/603–60 mos; male and femaleRacecadotril vs placeboDuration of diarrhea (65% reduction)10Lehert P. Chéron G. Calatayud G.A. et al.Racecadotril for childhood gastroenteritis: an individual patient data meta-analysis.Dig Liver Dis. 2011; 43: 707-713Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar Open table in a new tab In the current study from Bangladesh, the investigators evaluated the effects of a novel anti-rotaviral protein (ARP1), which consisted of the variable domains of llama heavy chain (VHH) antibodies expressed in yeast or placebo, on the severity, duration, and fecal excretion of rotavirus in 176 male infants with severe rotavirus gastroenteritis. The infants were given ARP1 or a maltodextrin placebo after admission for 4 or 5 days at a dose of 15–30 mg/kg body weight. Among children who had rotavirus infection alone, active treatment resulted in a significant decrease in the cumulative stool output, a reduction in overall stool frequency, and a more rapid decrease in frequency; in contrast, no improvements were seen among children who were also infected with other organisms and neither group experienced a change in the duration of diarrhea or vomiting, or excretion of rotavirus or rotavirus-specific immunoglobulin A levels.4Sarker S.A. Jäkel M. Sultana S. et al.Anti-rotavirus protein reduces stool output in infants with diarrhea: a randomized placebo-controlled trial.Gastroenterology. 2013; 145: 740-748Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar This study highlights some of the challenges of conducting studies on gastroenteritis in young children. In defining the outcomes to be measured, most investigators choose the parameters used by Sarker et al, the severity of diarrhea (which can be estimated by stool volume or output), stool frequency, duration of diarrhea or time to resolution, amount of oral or intravenous rehydration fluid required per kilogram of body weight, and resolution of associated symptoms such as vomiting. The illness can be severe, but with appropriate rehydration, can resolve rapidly, with most illnesses resolving in 2–3 days. Several studies, particularly those studying vaccine efficacy, have used a scoring system to evaluate the severity of diarrhea at presentation. The most widely used system is the 20-point Vesikari scoring system,11Ruuska T. Vesikari T. Rotavirus disease in Finnish children: use of numerical scores for clinical severity of diarrhoeal episodes.Scand J Infect Dis. 1990; 22: 259-267Crossref PubMed Scopus (573) Google Scholar although others have been described.12Givon-Lavi N. Greenberg D. Dagan R. Comparison between two severity scoring scales commonly used in the evaluation of rotavirus gastroenteritis in children.Vaccine. 2008; 26: 5798-5801Crossref PubMed Scopus (50) Google Scholar The investigators did not use a scoring system to evaluate diarrheal severity, but did recruit only dehydrated children who would be expected to have at least moderate to severe diarrhea. Nonetheless, it would have been useful to be able to stratify children with a severity score to understand whether the ARP1 was more effective in milder or, as expected, more severe disease. In evaluating the efficacy of an intervention against diarrhea, consideration must also be given to what constitutes a clinically significant reduction in duration, with most studies aiming at a reduction of ≥12 or ≥24 hours. Young children can also pass soft or unformed stool even when the diarrhea has settled, making the determination of the end of an episode challenging. A reduction in duration in hours can be difficult to measure, unless the sample size is large. Reduction in stool weight can be a more objective measure of resolution, but because of the problem of mixing of stool and urine in young children, these estimations are best done in male infants, as in the study reported here. However, although stool output and frequency decreased in children given ARP1, other parameters were not different. These results indicate that ARP1 does have some effect on rotavirus gastroenteritis, a finding supported by previous animal and in vitro studies conducted by these and other investigators.13Aladin F. Einerhand A.W. Bouma J. et al.In vitro neutralisation of rotavirus infection by two broadly specific recombinant monovalent llama-derived antibody fragments.PLoS One. 2012; 7: e32949Crossref PubMed Scopus (31) Google Scholar, 14Vega C.G. Bok M. Vlasova A.N. et al.Recombinant monovalent llama-derived antibody fragments (VHH) to rotavirus VP6 protect neonatal gnotobiotic piglets against human rotavirus-induced diarrhea.PLoS Pathog. 2013; 9: e1003334Crossref PubMed Scopus (53) Google Scholar, 15van der Vaart J.M. Pant N. Wolvers D. et al.Reduction in morbidity of rotavirus induced diarrhoea in mice by yeast produced monovalent llama-derived antibody fragments.Vaccine. 2006; 24: 4130-4137Crossref PubMed Scopus (89) Google Scholar Given that ARP1 is a specific antibody fragment derived from a VHH antibody raised against a G3P[3] virus, which seems to be effective in children infected with human viruses of differing genotypes, it is important to understand the mechanism by which it exerts its action. ARP1 is a yeast-expressed variable antigen-binding molecule derived from a new type of antibody, described first in the sera of dromedaries in 1989 and now known in all species of the Camelidae family. These antibodies are heavy chain only and lack the first constant heavy chain; compared with human heavy chain variable regions, VHH often have a longer CDR3 loop, which results in an increased surface area and antigen-binding repertoire.16Vu K.B. Ghahroudi M.A. Wyns L. et al.Comparison of llama VH sequences from conventional and heavy chain antibodies.Mol Immunol. 1997; 34: 1121-1131Crossref PubMed Scopus (250) Google Scholar They are heat and acid stable and have increased solubility, making it possible to consider them in a range of therapeutic and diagnostic applications, including their use as functional foods, given the ability to express them in large quantities at low cost.17Vanlandschoot P. Stortelers C. Beirnaert E. et al.Nanobodies®: new ammunition to battle viruses.Antiviral Res. 2011; 92: 389-407Crossref PubMed Scopus (107) Google Scholar Although this was not investigated in the trial, previous studies have shown that ARP1 or similar proteins can bind and neutralize a range of animal and human rotavirus strains in vitro and reduce the severity of rotavirus diarrhea in mouse pups, but the exact site of binding or interaction is not known.13Aladin F. Einerhand A.W. Bouma J. et al.In vitro neutralisation of rotavirus infection by two broadly specific recombinant monovalent llama-derived antibody fragments.PLoS One. 2012; 7: e32949Crossref PubMed Scopus (31) Google Scholar, 15van der Vaart J.M. Pant N. Wolvers D. et al.Reduction in morbidity of rotavirus induced diarrhoea in mice by yeast produced monovalent llama-derived antibody fragments.Vaccine. 2006; 24: 4130-4137Crossref PubMed Scopus (89) Google Scholar A similar heavy chain antibody directed against the rotavirus group–specific antigen VP6 derived from a bovine strain, which used baculovirus expression vectors and insect larvae as living biofactories, showed that anti-VP6 VHH antibodies protected neonatal gnotobiotic piglets against human rotavirus infection.12Givon-Lavi N. Greenberg D. Dagan R. Comparison between two severity scoring scales commonly used in the evaluation of rotavirus gastroenteritis in children.Vaccine. 2008; 26: 5798-5801Crossref PubMed Scopus (50) Google Scholar It is possible that ARP1 also binds VP6, the most abundant and immunodominant rotavirus protein, because the small antibody fragments may be able to pass through the outer coat, which has the VP7 and VP4 protein to access exposed VP6 binding sites on the middle coat.18Garaicoechea L. Olichon A. Marcoppido G. et al.Llama-derived single-chain antibody fragments directed to rotavirus VP6 protein possess broad neutralizing activity in vitro and confer protection against diarrhea in mice.J Virol. 2008; 82: 9753-9764Crossref PubMed Scopus (96) Google Scholar If the mechanism of action is virus specific, it should result in a decrease in viral replication, but this was not measurable by an enzyme-linked immunosorbent assay–based antigen shedding evaluation. The study results show limited effect, but seem promising. Vaccines, even when effective in the majority of children, take time to induce an immune response, whereas VHH fragments can be immediately protective at a mucosal surface. However, there are several questions that must be answered before ARP1 or other VHH molecules can proceed to clinical use. A critical point to be considered is the host's immune response to a foreign protein. Although only 1 child reported a rash and an earlier animal study reported no detectable host antibodies,4Sarker S.A. Jäkel M. Sultana S. et al.Anti-rotavirus protein reduces stool output in infants with diarrhea: a randomized placebo-controlled trial.Gastroenterology. 2013; 145: 740-748Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 14Vega C.G. Bok M. Vlasova A.N. et al.Recombinant monovalent llama-derived antibody fragments (VHH) to rotavirus VP6 protect neonatal gnotobiotic piglets against human rotavirus-induced diarrhea.PLoS Pathog. 2013; 9: e1003334Crossref PubMed Scopus (53) Google Scholar careful assessment of a specific immune response and monitoring of outcomes are essential for evaluation of safety of this biological therapy. In addition, an understanding of the mechanism of action, in terms of the site of action and of the functional modifications that decrease pathogenesis of gastroenteritis, are needed. Nonetheless, the biological engineering that resulted in the production of VHH antibodies, also called nanobodies, has led to the consideration of new modalities for therapy, diagnostics, and imaging.17Vanlandschoot P. Stortelers C. Beirnaert E. et al.Nanobodies®: new ammunition to battle viruses.Antiviral Res. 2011; 92: 389-407Crossref PubMed Scopus (107) Google Scholar, 19Virdi V. Coddens A. De Buck S. et al.Orally fed seeds producing designer IgAs protect weaned piglets against enterotoxigenic Escherichia coli infection.Proc Natl Acad Sci U S A. 2013; 110: 11809-11814Crossref PubMed Scopus (94) Google Scholar Careful clinical evaluation in other settings will be necessary to measure safety and efficacy of a potentially widely applicable tool. Anti-Rotavirus Protein Reduces Stool Output in Infants With Diarrhea: A Randomized Placebo-Controlled TrialGastroenterologyVol. 145Issue 4PreviewRotavirus infection is a leading cause of morbidity and mortality in children younger than 5 years of age. Current treatment options are limited. We assessed the efficacy of a llama-derived, heavy-chain antibody fragment called anti-rotavirus protein (ARP1), in modifying the severity and duration of diarrhea in male infants with rotavirus infection. Full-Text PDF