Paediatric human metapneumovirus infection: Epidemiology, prevention and therapy

Journal of Clinical Virology 59 (2014) 141–147

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Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv

Review

Paediatric human metapneumovirus infection: Epidemiology, prevention and therapy Nicola Principi, Susanna Esposito ∗ Pediatric High Intensity Care Unit, Department of Pathophysiology and Transplantation, Università degli Studi di Milano, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy

a r t i c l e

i n f o

Article history: Received 19 November 2013 Received in revised form 27 December 2013 Accepted 5 January 2014 Keywords: Antiviral therapy Human metapneumovirus Monoclonal antibodies Respiratory infection Respiratory viruses Vaccines

a b s t r a c t Since its discovery in 2001, human metapneumovirus (hMPV) has been identified as one of the most frequent causes of upper and lower respiratory tract infections. Although a considerable number of hMPV infections are diagnosed in adults and the elderly, the highest incidence of infection is among children as seropositivity for hMPV approaches 100% by 5–10 years of age. Most of the diseases due to hMPV are mild or moderate, tend to resolve spontaneously, and only require outpatient treatment. However, some may be severe enough to require hospitalisation or, albeit rarely, admission to a paediatric intensive care unit because of acute respiratory failure. Mortality is exceptional, but may occur. The most severe diseases generally affect younger patients, prematurely born children, and children who acquire nosocomial hMPV infection and those with a severe chronic underlying disease. Global hMPV infection has a major impact on national health systems, which is why various attempts have recently been made to introduce effective preventive and therapeutic measures; however, although some are already in the phase of development (including vaccines and monoclonal antibodies), there is currently no substantial possibility of prevention and, despite its limitations, ribavirin is still the only possible treatment. Given the risk of severe disease in various groups of high-risk children and the frequency of infection in the otherwise healthy paediatric population, there is an urgent need for further research aimed at developing effective preventive and therapeutic measures against hMPV. © 2014 Elsevier B.V. All rights reserved.

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epidemiology of hMPV infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Prevention of hMPV infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.1. Live attenuated vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.2. Subunit vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.3. Virus-like particle vaccines (VLPVs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Monoclonal antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Antiviral therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Nucleoside analogues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Fusion inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Competing interests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ethical approval . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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∗ Corresponding author at: Pediatric High Intensity Care Unit, Department of Pathophysiology and Transplantation, Università degli Studi di Milano, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Via Commenda 9, 20122 Milano, Italy. Tel.: +39 02 55032498; fax: +39 02 50320206. E-mail address: [email protected] (S. Esposito). 1386-6532/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jcv.2014.01.003

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1. Introduction Since its discovery in 2001 [1], human metapneumovirus (hMPV) has been identified as one of the most frequent causes of upper (URTI) and lower respiratory tract infection (LRTI), with a disease spectrum that is similar to that of respiratory syncytial virus (RSV) [2]. Although a considerable number of hMPV infections are diagnosed in adults and the elderly, the highest incidence of infection is among children [3,4] as seropositivity for hMPV approaches 100% by 5–10 years of age [3]. Most of the diseases due to hMPV are mild or moderate, tend to resolve spontaneously, and only require outpatient treatment. However, some may be severe enough to require hospitalisation or, albeit rarely, admission to a paediatric intensive care unit (ICU) because of acute respiratory failure [4–17]. Mortality is exceptional and may occur in 5–10% of hMPV-positive children admitted to the ICU [4–17]. This means that global hMPV infection has a major impact on national health systems, which is why various attempts have recently been made to introduce effective preventive and therapeutic measures. The main aim of this paper is to review the attempts made to protect and treat humans against hMPV infection, and to discuss which children may benefit most from the availability of more effective measures.

2. Epidemiology of hMPV infection A recent evaluation of the total burden of hMPV infection in children living in the USA has found that, every year (mainly during the winter), hMPV leads to a number of hospitalisations and outpatient clinic and emergency department visits that is quite similar to that due to influenza viruses [17]. HMPV was detected in 6% of hospitalised children, 7% of those seen in outpatient clinics, and 7% of those examined in emergency departments, with the highest incidence rates being observed among children in the first months of life. Overall, annual rates of hospitalisation associated with hMPV infection were 1 per 1000 children less than 5 years of age, 3 per 1000 infants less than 6 months of age, and 2 per 1000 children 6–11 months of age. Children hospitalised with hMPV infection, as compared with those hospitalised without hMPV infection, were older and more likely to receive a diagnosis of pneumonia or asthma, to require supplemental oxygen, and to have a longer stay in the intensive care unit. The estimated annual burden of outpatient visits associated with hMPV infection was 55 clinic visits and 13 emergency department visits per 1000 children. The majority of hMPV-positive inpatient and outpatient children had no underlying medical conditions, although premature birth and asthma were more frequent among hospitalised children with hMPV infection than among those without hMPV infection. These data show that hMPV infection is associated with a substantial burden of hospitalisations and outpatient visits among children throughout the first 5 years of life and most children with hMPV infection were previously healthy [17]. The finding that the risk of severe hMPV infection is greater in younger subjects was previously reported by Mullins et al. [18] and Papenburg et al. [19]. However, this does not mean that older children are not at risk of developing severe hMPV infection: Spaeder et al. analysed the demographic and clinical data, and associated morbidity and mortality outcomes, of 111 hMPV-infected children admitted to an ICU because of severe respiratory problems [20], and found that 54% were aged <2 years, but as many as 26% were aged ≥5 years. Similarly, Eggleston et al. found that the mean age of 26 children admitted to an ICU because of hMPV infection was 3.43 years, and that 34.6% were aged ≥5 years [21]. Together with a younger age, prematurity and the nosocomial acquisition of viral infection have almost systematically been found to be risk factors for severe hMPV infection associated with acute respiratory failure [17–21].

Table 1 Risk factors for severe paediatric human metapneumovirus infection. Characteristic

Risk factor

Age Gestational age Acquisition of infection Underlying disease

<2 years <37 weeks Nosocomial Presence of chronic pulmonary disease (including asthma), congenital heart disease, neuromuscular disorders, trisomy 21, or congenital or acquired immunodeficiency

However, the factor most clearly associated with more severe hMPV infection is the presence of an underlying severe chronic disease. Edwards et al. found that 40% of the children hospitalised because of hMPV-related respiratory problems had underlying high-risk conditions, including asthma and chronic lung disease, whereas only 22% of the outpatients with hMPV infection had a chronic clinical problem [17]. Spaeder et al. found that 59% of the paediatric patients admitted to an ICU with laboratoryconfirmed hMPV infection showed severe respiratory involvement, with chronic lung and neuromuscular diseases being among the most frequent [20]. In their study, there were 111 patients with laboratory-confirmed hMPV admitted to an ICU: the median hospital length of stay was 7 days (interquartile range 4–18 days) and median ICU length of stay was 4 days (interquartile range 1–10 days). Ten patients (9%) did not survive to discharge. Adjusting for female gender, chronic medical conditions, hospital acquisition of infection and severity of illness score, logistic regression analysis demonstrated that female gender, hospital acquisition of infection, and chronic medical conditions each independently increased the odds of mortality (odds ratios 14.8, 10.7, and 12.7, respectively). Hahn et al. found that 68% of 238 children hospitalised because of hIMPV infection had at least one underlying medical condition, with chronic pulmonary disease, congenital heart disease, neuromuscular disorders and trisomy 21 being the most frequently associated with hMPV-related acute respiratory failure [22]. Immunocompromising clinical problems such as cancer or hematopoietic stem cell transplantation (HSCT) are documented risk factors in adults [23,24] and, although there are few published data concerning children with these conditions, some case reports of fatal or very severe pneumonia in immunocompromised paediatric patients suggest that a significant reduction in the efficiency of the immune system may also be a risk factor for the development of severe hMPV infection during the first years of life [24–26]. A higher viral load can condition the severity of hMPV infection as Bosis et al. found significantly higher hMPV loads in hospitalised children and those with LRTIs than in outpatients with URTIs [27]. Genetic analysis of hMPV isolates has revealed two major groups (A and B) and four minor sub-groups (A1, A2, B1 and B2), mainly based on the sequence variability of the attachment (G) and fusion (F) surface glycoproteins [28]. The existence of two further subgroups, A2a and A2b, has also been suggested [29,30]. Multiple lineages can circulate each year and predominant circulating hMPV lineages vary by year [30]. In several studies, hMPV-F appeared highly conserved, whereas hMPV-G exhibited greater diversity [30,31]. According to some authors, the highly conserved F protein constitutes an antigenic determinant that mediates crosslineage neutralisation and protection [32], while other studies have reported a difference in reactivity between the two genotypes [33]. In conclusion, the available epidemiological data indicate that hMPV infection is common at all paediatric ages, but is more likely to be severe in younger patients, prematurely born children, children acquiring nosocomial hMPV infection, and those with severe chronic underlying diseases (Table 1). These are therefore the subjects that may receive the greatest benefit from effective preventive and therapeutic measures although, given the

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frequency of the infection in otherwise healthy children during winter, universal prevention cannot be excluded. 3. Prevention of hMPV infection As shown in Table 2, current research on prevention of hMPV infection is currently based on the development of vaccines and monoclonal antibodies therapies. 3.1. Vaccines The finding that cynomolgus macaques inoculated with hMPV three times in 10 weeks were not protected against challenge infection eight months after the last inoculation suggests that wild-type hMPV infection induces only transient protective immunity [34]. Furthermore, adults and older children are frequently re-infected by other Paramyxoviruses because natural RSV or parainfluenza virus (PIV) infections do not induce lifelong protection. This suggests that, in order to be really effective, a vaccine against hMPV should be more immunogenic and protective than natural infection. Various vaccine strategies have been investigated, but only liveattenuated vaccines (LAVs), subunit vaccines, and vaccines based on virus-like particles (VLPVs) are currently in a development phase. Research into inactivated vaccines for all Paramyxoviruses has been abandoned mainly because the use of a formalininactivated hMPV vaccine led to an increase in lung diseases in animal models and a change in cytokine profiles indicating a hypersensitive response [35,36], a problem that had been previously observed when a formalin-inactivated RSV vaccine was used in children [37]. However, none of the vaccines under development has yet been licensed, and only some are supported by evidence that is strong enough to justify clinical trials in humans. 3.1.1. Live attenuated vaccines LAVs have the theoretical advantage of mimicking natural infection and inducing an immune response that is similar to that induced by the wild virus. In order to be administered without problems, LAVs should be adequately attenuated and there should be little risk of reversion or the recovery of viral pathogenicity. The first LAV to be described was a preparation in which a chimeric bovine PIV type 3 harbouring the F and hemagglutininneuraminidase genes of human PIV type 3 was used as a vector to express the F protein of a group A hMPV strain. The immunisation of experimental animals induced neutralising antibodies against both hMPV groups, although the levels of antibodies against heterotopic group B strains were significantly lower [38]. Vaccinated African green monkeys were protected against a challenge with the homologous hMPV strain, which led to undetectable viral titres in the lower respiratory tract (LRT) and a more than 100-fold reduction in the upper respiratory tract (URT) [38]. A second group of LAVs was obtained by deleting non-essential hMPV genes, such as those of the small hydrophobic (SH) attachment (G) or second open reading frame of M2 (M2-2) proteins [39–41]. After topical administration in the respiratory tract of African green monkeys the replication of the SH virus was only slightly less efficient than that of wild-type hMPV, thus suggesting that this agent was not a good vaccine candidate [39–41]. On the contrary, G and M2-2 viruses were respectively reduced 6- and 160-fold in the URT, and 3200- and 4000-fold in the LRT, with marginal virus shedding [39–41]. Each of these gene-deleted viruses was highly immunogenic and protective against a wild-type hMPV challenge, and are thus considered adequately attenuated and promising vaccine candidates. Another recombinant hMPV vaccine was produced by generating temperature-sensitive hMPVs [42]. Repeated low-temperature

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passages of wild-type hMPV in Vero cells led to the accumulation of mutations in the viral genome which, when inserted into a recombinant hMPV by means of reverse genetics, produced a temperature-sensitive phenotype. The replication of these temperature-sensitive viruses was reduced in the URT and undetectable in the LRT of hamsters, but protective titres of hMPVspecific antibodies were induced and, after immunisation, the LRT was completely protected against a challenge infection with a heterologous hMPV strain, and viral titres in the URT were reduced 10,000-fold [43]. However, the duration of the protection was relatively limited, although immunisation primed a good secondary hMPV specific lymphoproliferative response after a challenge infection. Replacing the nucleoprotein (N) or phosphoprotein (P) of hMPV by their avian MPV type C counterparts led to the development of chimeric viruses capable of inducing high levels of protective neutralising antibodies in hamsters, although low replication levels in the trachea and nasopharynx of treated African green monkeys indicated significant attenuation in comparison with wild-type hMPV [44]. As the available data suggest that the immunogenicity and protective efficacy of both chimerae are comparable with those of wild-type hMPV, a phase one study of the use of an hMPVP virus-based vaccine in seropositive children is currently ongoing [45].

3.1.2. Subunit vaccines These vaccines are easier to produce than LAVs and avoid the problem of attenuation. Vaccines against hMPV are mainly based on the F protein, and Herfst et al. have evaluated the immunogenicity and efficacy of an iscom matrix-adjuvanted hMPV F protein subunit vaccine (Fsol) in cynomolgus macaques [43], finding that it induced hMPV F-specific antibody responses, virus neutralising antibody titres, and cell immune responses. Unfortunately, the rapid waning of the induced humoral immune response was considered an obstacle for further development. A second subunit vaccine was prepared using F protein lacking the transmembrane domain (FTM). Cseke et al. immunised cotton rats with control vector, F alone, F followed by FTM, or FTM alone on day 0 and 14, and then challenged them with live hMPV on day 28 [46]. All of the three groups that received some form of hMPV F immunisation mounted neutralising antibody responses and showed partial protection against virus shedding in the lungs, with the FTM-immunised animals showing the greatest degree of protection (a >1500-fold reduction in lung virus titres). All three groups also showed a slight reduction in nasal virus shedding, and there was no evidence of a Th2-type response or increased lung disease.

3.1.3. Virus-like particle vaccines (VLPVs) VLPVs mimic the structure of the surface of virus particles and can be used as carriers of foreign antigens. A vaccine incorporating hMPV glycoproteins from either of the two main lineages in retroviral core particles that functionally display F and G hMPV surface proteins [47] induced a strong humoral immune response against homologous and heterologous strains when injected in mice. The induced neutralising antibodies prevented mortality upon subsequent lung infection lungs with homologous and heterologous viruses, and the viral titres in the lungs of the immunised animals were significantly lower than those of control animals. On the basis of these data, it was concluded that a hMP VLPV that induces crossprotective immunity in mice is a promising approach to preventing hMPV infections [47].

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Table 2 Products under development for the prevention of human metapneumovirus (hMPV) prevention and results of available research. Preventive approach

Product

Vaccines

Live attenuated vaccines Chimeric bovine PIV type 3 harbouring the F and hemagglutinin-neuraminidase genes of human PIV type 3 used as a vector to express the F protein of a group A hMPV strain Vaccines lacking the small hydrophobic (SH) attachment (G) and the second open reading frame of M2 (M2-2) proteins Obtained by means of repeated low-temperature passages of wild-type hMPV in Vero cells Characterised by chimeric viruses in which the nucleoprotein (N) or phosphoprotein (P) of hMPV was replaced by their avian MPV type C counterparts

Main results

Subunit vaccines Iscom matrix-adjuvanted hMPV F protein subunit vaccine (Fsol) F protein lacking the transmembrane domain (FTM) vaccine

Monoclonal antibodies

Virus-like particle vaccines Incorporation of hMPV glycoproteins from either of the two main lineages into retroviral core particles MAb 338

MPE8

Immunisation of experimental animals induces neutralising antibodies against both groups of hMPV

Following topical administration in the respiratory tract of African green monkeys, each gene-deleted virus was highly immunogenic and protective against a wild-type hMPV challenge Protective titres of hMPV-specific antibodies induced in experimental animals, although the duration of protection was relatively limited Capable of inducing high levels of protective neutralising antibodies in hamsters, although significantly attenuated in comparison with wild-type hMPV; a phase one study of an hMPV-P virus-based vaccine in seropositive children is currently ongoing Induced hMPV F-specific antibody responses, virus neutralising antibody titres, and cell immune responses, although the induced humoral immune response rapidly waned Cotton rats challenged intranasally mounted neutralising antibody responses and showed partial protection against virus shedding in the lungs When injected into mice, induced a strong humoral immune response against both homologous and heterologous strains Capable of neutralising strains from the four hMPV subgroups, decreasing lung viral titres in hamsters when administered 24 h before intranasal infection, and reducing hMPV replication when administered 48 h after infection Did not lead to the selection of viral escape mutants that evade antibody targeting, and showed potential prophylactic efficacy in animal models of hMPV infection

PIV: parainfluenza virus.

3.2. Monoclonal antibodies Palivizumab is a humanised monoclonal antibody against the RSV fusion (F) protein that is very effective in neutralising the virus and is currently used as a prophylactic agent in high-risk infants during the RSV season [48]. The F proteins of RSV and hMPV are relatively similar and, although palivizumab is not effective against hMPV [49], it was hypothesised that a monoclonal antibody against the hMPV F protein would be prophylactic and perhaps also therapeutic. This was confirmed when it was demonstrated that MAb 338, one of a number of monoclonal antibodies generated by Ulbrandt et al. [50], neutralised strains from the four hMPV subgroups, and decreased lung viral titres when administered to hamsters 24 h before intranasal infection [51]. The prophylactic and therapeutic benefit of MAb 338 was later supported by data collected by Hamelin et al., who found that it could reduce hMPV replication even when administered 48 h after infection [52,53]. When screening blood donors, Corti et al. identified a human monoclonal antibody (MPE8), specific for the pre-fusion F protein that potentially cross-neutralised RSV and hMPV, as well as bovine RSV and pneumonia virus of mice (PVM) [54]. It did not lead to the selection of viral escape mutants that evade antibody targeting, and was potentially prophylactically efficacious in animal models of RSV and hMPV infection; furthermore, it was also prophylactically and therapeutically efficacious in the more relevant model of lethal PVM infection, thus suggesting its possible use in humans.

3.3. Conclusions The studies of possible preventive measures indicate that none of the hMPV vaccines is ready for use in humans, and several years of intensive research will be needed before one becomes available. Furthermore, despite promising preliminary results, monoclonal

antibodies targeting the hMPV F protein have not yet entered clinical trials. 4. Antiviral therapy Table 3 shows the main antiviral drugs against hMPV that have so far been tested, but all of them are still a long way from the clinical trial stage. Table 3 Antiviral drugs against human metapneumovirus (hMPV). Antiviral class

Product

Main results

Nucleoside analogues Fusion inhibitors

Ribavirin

Debatable efficacy after aerosol, oral or intravenous administration HRA2 had very potent activity against all four hMPV subgroups in BALB/c mice; pulmonary inflammation, levels of pro-inflammatory cytokines/chemokines, and airway obstruction were also significantly decreased in HRA2-treated mice SiRNA45 targets the nucleoprotein messenger RNA, and has inhibitory concentration 50 (IC50) values of <0.078 nM against representative strains from the four hMPV subgroups, whereas siRNA60, which targets the phosphoprotein mRNA, had IC50 values of between 0.090 and <0.078 nM against the same panel of hMPV strains; in a murine model, the prophylactic administration of an EvaderTM Dicer-substrate siRNA was effective at partially inhibiting viral replication of hMPV, with inhibition achieved without inducing cytokines or off-target effects

Peptides derived from the heptad repeat A and B (HRA and HRB) domains of the hMPV fusion protein

Small interfering RNAs (siRNAs)

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4.1. Nucleoside analogues Ribavirin is a nucleoside analogue that is effective against other Paramyxoviruses in vitro and in vivo [55], and theoretically could be used to treat hMPV infection. It disrupts viral purine metabolism and inhibits viral RNA polymerase, and has good in vivo activity against experimental hMPV infection [56]. It also up-regulates CD4 and CD8 T lymphocyte-derived interleukin (IL)-2, tumour necrosis factor-␣, and interferon-␥, and down-regulates T helper 2 cytokines such as IL-10, which suggests an immunomodulatory effect that can increase the containment of viral infection [57]. However, there are few and conflicting data regarding its efficacy against hMPV infection in humans, probably because the best route of administration and the most effective dose have not yet been defined. Inhaled ribavirin reduces severe RSV infection in infants and young children and, as in vitro studies have shown that similar concentrations of ribavirin inhibit hMPV, it could be administered by aerosol at the dose usually recommended to treat RSV [58]. However, widespread use of the aerosolised drug has been impeded by its high cost, its teratogenicity in healthcare workers, and its potential for deteriorating respiratory function [59]. The bioavailability of oral ribavirin varies and the optimal oral dose for patients with Paramyxovirus infection has not yet been established [60]. Intravenous (i.v.) ribavarin has been tried in a few patients with hMPV disease at doses whose efficacy, safety and tolerability have never been evaluated in controlled clinical trials involving children, but its efficacy is debatable [26]. Dokos et al. found that i.v. ribavirin combined with i.v. immunoglobulins (IVIG) and corticosteroids had no effect on a 10-year-old girl with chronic graft-versus-host disease following allogeneic HSCT [26]. The results obtained by Park et al. in a study of adult patients with haematological disease and Paramyxovirus infection (including 21 cases of hMPV infection) were also negative [61]. One hundred and fourteen of the studied patients (78%) received oral ribavirin and the remaining 31 (21%) did not, and the 30-day mortality and underlying respiratory death rates were respectively 31% (35/114) and 12% (14/114) in the ribavirin group, and 19% (6/31) and 16% (5/31) in the non-ribavirin group (p = 0.21 and p = 0.56). In the hPMV case–control study, the 30-day mortality rate was 24% in the ribavirin group (5/21) and 19% (4/21) in the non-ribavirin group (p = 0.71) [61]. In addition, logistic regression with inverse probability of treatment weighting using propensity scores revealed no significant difference in 30-day mortality (adjusted hazard ratio of 1.3, 95% confidence interval [95% CI] 0.3–5.8) between the two groups. Steroid use (adjusted odds ratio, 5.67; p = 0.01) and URTI (adjusted odds ratio, 0.07; p = 0.001) were independently associated with mortality [61]. Completely different results have been obtained by other authors. There are reports regarding the successful use of i.v. ribavirin in combination with IVIG in treating hMPV pneumonia in one adult who had undergone a lung transplant [62] and another who had undergone HSCT [63]. Moreover, oral and aerosolised ribavirin together with IVIG was found to be effective in two adults with pneumonia and HSCT [64], and i.v. ribavirin with IVIG was effective in a 4-year-old girl with pneumonia and acute lymphoblastic leukaemia [65]. Moreover, two cases of pneumonia in children with Burkitt lymphoma had a favourable evolution after the combined administration of oral ribavirin and IVIG [25,66]. No other nucleoside analogue that theoretically active against hMPV is currently in an advanced phase of development. 4.2. Fusion inhibitors It has been suggested that peptides capable of inhibiting the fusion of viral and cell membranes interacting with the hMPV F protein could play a role in reducing viral replication and the

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development of infection. Deffrasnes et al. have assessed the inhibitory potential of a number of peptides derived from the heptad repeat A and B (HRA and HRB) domains of the hMPV fusion protein [67], and one (HRA2) had very potent activity against hMPV. BALB/c mice that simultaneously received HRA2 and a lethal hMPV intranasal challenge were completely protected from clinical symptoms and mortality. On day 5 post-infection, the HRA2-treated mice had undetectable lung viral loads that were significantly lower than those found in the untreated mice (3 × 104 50% tissue culture infective doses/lung) [67]. Pulmonary inflammation, the levels of pro-inflammatory cytokines/chemokines (i.e. RANTES, interferon-␥ and monocyte chemoattractant protein 1), and airway obstruction were also significantly decreased in the HRA2-treated mice. Further attempts to find molecules capable of interfering with hMPV replication have been made using small interfering RNAs (siRNAs), a class of 20/25-nucleotide double-stranded RNA molecules involved in the RNA interference pathway that can modify the expression of a specific gene, and two highly potent siRNAs were identified as having subnanomolar 50% inhibitory concentrations (IC50) [68]. SiRNA45 targets the nucleoprotein messenger RNA (mRNA) and had an IC50 of <0.078 nM against representative strains from the four hMPV subgroups, whereas siRNA60, which targets the phosphoprotein mRNA, had IC50 values of between 0.090 and <0.078 nM against the same panel of hMPV strains. Darniot et al. showed that in a murine model the prophylactic administration of an EvaderTM Dicer-substrate siRNA was effective at partially inhibiting viral replication of hMPV (13 × 103 versus 29 × 103 PFU/g of lung; p < 0.01), which was not the case for the control, a mismatched Dicer-substrate siRNA [69]. Inhibition was achieved without inducing cytokines or off-target effects. This approach against hMPV is an important step in the development of synthetic siRNA as a therapeutic agent for this virus with inhibition achieved without inducing cytokines or off-target effects. 5. Conclusion The large amount of epidemiological data regarding hMPV infection in children illustrate its importance and the potential relevance of preventing it, at least in younger children, those born prematurely and those with a severe chronic underlying disease. Unfortunately, there is currently no substantial possibility of prevention and, despite its limitations, ribavirin is still the only possible treatment. Given the risk of severe disease in various groups of high-risk children and the frequency of infection in the otherwise healthy paediatric population, there is an urgent need for further research aimed at developing effective preventive and therapeutic measures against hMPV. Funding This review was supported by a grant from the Italian Ministry of Health (Bando Giovani Ricercatori 2009). Competing interests None. Ethical approval Not required. References [1] van den Hoogen BG, de Jong JC, Groen J, Kuiken T, de Groot R, Fouchier RA, et al. A newly discovered human pneumovirus isolated from young children with respiratory tract disease. Nat Med 2001;7:719–24.

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