1 MedImmune Vaccines Inc., 297 North Bernardo Avenue, Mountain View, CA 94043, USA
2 Department of Virology, Erasmus Medical Center, Rotterdam, The Netherlands
Correspondence
Aurelia A. Haller
aurelia.haller{at}globeimmune.com
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ABSTRACT |
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Present address: GlobeImmune Inc., Aurora, CO 80010, USA.
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INTRODUCTION |
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On the basis of electron microscopy and comparison of viral genome sequence organization, hMPV was assigned to the Metapneumovirus genus of the Paramyxoviridae family. hMPV contains a non-segmented, negative-sense RNA genome approximately 13 370 nucleotides in length (van den Hoogen et al., 2002; Biacchesi et al., 2003
). The genomic organization for hMPV is similar but not identical to that of RSV. hMPV harbours open reading frames (ORFs) for at least eight viral proteins (3'-N-P-M-F-M2-SH-G-L-5'). However, hMPV lacks the non-structural proteins NS1 and NS2 of RSV, and the gene order of RSV and hMPV differs significantly. In hMPV, the M2-1 and small hydrophobic (SH) protein ORFs are located between the fusion (F) and glycoprotein (G) genes, which is unlike the RSV genomic organization. The M2-1 protein of RSV promotes processive RNA synthesis and readthrough at RSV gene junctions. Future studies will determine whether the M2-1 protein of hMPV plays a similar role in hMPV replication. Deletion of a number of RSV genes such as M2-2, SH, G, NS1 and NS2 was not deleterious to the virus and such RSV deletion mutants have been evaluated in primates as putative live attenuated vaccine candidates (Jin et al., 2003
). Similar strategies will be employed to generate live attenuated hMPV vaccine candidates once a reverse genetics system is established for hMPV. Two subgroups of hMPV (subgroups A and B) were identified based on sequence comparison of the F and G genes derived from a number of different clinical isolates. Within each subgroup, two genetic sublineages were categorized, A1 and A2 for hMPV subgroup A, and B1 and B2 for hMPV subgroup B. The F genes of subgroups A and B are highly conserved and display >95 % identity at the amino acid level. In contrast, the hMPV G proteins are variable and show only 35 % amino acid identity (van den Hoogen et al., 2004
; Biacchesi et al., 2003
). The F protein of hMPV/NL/1/00 displays a high degree of conservation with the F protein of a related metapneumovirus, avian pneumovirus subgroup C (APV C), a fowl pathogen. Avian pneumovirus causes swollen head syndrome in chickens and rhinotracheitis in turkeys (Buys et al., 1989
). In particular, the ectodomains of the F proteins of APV C and hMPV are closely related. hMPV may have evolved from the avian metapneumovirus, although hMPV is not infectious for birds (van den Hoogen et al., 2001
). Efforts to generate live attenuated hMPV vaccines, as well as to prepare neutralizing hMPV mAbs, have been initiated by a number of research institutions.
We recently reported the generation of a putative hMPV vaccine candidate, a recombinant live attenuated bovine/human PIV3 expressing the hMPV F protein (Tang et al., 2003). In the future, a plethora of putative hMPV vaccines, mAbs and antivirals will be generated, and animal models will be needed to evaluate these approaches for safety, efficacy and immunogenicity, where applicable. In this study, a number of small-animal and primate models were investigated to determine their ability to support hMPV replication in the respiratory tract and to produce an effective immune response. Mice, cotton rats, hamsters and ferrets were studied as small-animal models for hMPV replication, and rhesus monkeys and African green monkeys (AGMs) were tested as primate models. The results showed that Syrian golden hamsters, ferrets and AGMs were highly susceptible to hMPV infection and supported high levels of hMPV replication in the lower (LRT) and upper respiratory tract (URT). The data generated using these animal models will support licensing of hMPV vaccines, antivirals and prophylactic mAbs for high-risk children.
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METHODS |
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Small-animal studies.
Five-week-old hamsters (Mesocricetus auratus), BALB/c mice (Mus musculus), cotton rats (Sigmodon hispidus) (six animals per group) or ferrets (Mustela putorius) (four animals per group) were infected intranasally with 1x106 p.f.u. hMPV/NL/1/00. The animals were housed in individual micro-isolator cages. Four days post-infection, the nasal turbinates and lungs of the animals were harvested and homogenized. The titre of virus present in the tissues was determined by plaque assays on Vero cells, which were immunostained with hMPV polyclonal antisera. Ferrets were also monitored for changes in body temperature. For polyclonal antibody production, infected ferrets were maintained for 28 days post-infection at which time the animals were exsanguinated.
For the challenge studies, hamsters (18 animals per group) were infected intranasally with 1x105 p.f.u. hMPV/NL/1/00, hMPV/NL/1/99 or placebo medium (Opti-MEM) in a 0·1 ml volume. The different groups were maintained separately in micro-isolator cages. Four days post-infection, the nasal turbinates and lungs of six animals were harvested and homogenized. The titre of virus present in the tissues was determined by plaque assays on Vero cells by immunostaining with hMPV polyclonal antisera. Four weeks post-immunization, the remaining 12 animals were challenged intranasally with 1x106 p.f.u. hMPV/NL/1/00 (six animals) or 2x105 p.f.u. hMPV/NL/1/99 (six animals) in a 0·1 ml volume. Four days post-challenge, the nasal turbinates and lungs of the animals were collected and assayed for challenge virus replication by plaque assays on Vero cells. Plaques were visualized for quantification by immunostaining with hMPV polyclonal antisera.
Primate studies.
Three hMPV-seronegative and three RSV-seronegative AGMs (Cercopithecus aethiops) and four hMPV-seronegative rhesus monkeys (Macaca mulatta) (14 years old, 25 kg) were identified using an hMPV plaque reduction neutralization assay (PRNA) (described below) and an RSV F IgG ELISA (Immuno-Biological Laboratories), respectively, using primate pre-sera collected on day 18 prior to the study start date. The primates were housed in individual micro-isolator cages. The monkeys were anaesthetized with a ketamine/valium mixture and infected intranasally and intratracheally with hMPV/NL/1/00 or RSV A2. On day 1, each animal received a dose of 2 ml containing 1·3x105 p.f.u. hMPV ml1 or 3·5x105 p.f.u. RSV ml1. Nasopharyngeal (NP) swabs were collected daily for 11 days and tracheal lavage (TL) specimens were collected on days 1, 3, 5, 7 and 9 post-immunization. Blood samples for serological assays were collected on days 1, 7, 14, 21 and 28. The animals were monitored for body temperature changes indicating a fever, signs of a cold, runny nose, sneezing, loss of appetite and change in body mass. hMPV or RSV present in the primate NP and TL specimens was quantified by plaque assay using Vero cells. Mean peak virus titres represent the mean of the peak virus titre measured for each animal on any of the 11 days following immunization.
Plaque reduction neutralization assay.
PRNAs were carried out for sera obtained on days 1 and 28 post-infection from hamsters and primates infected with hMPV/NL/1/00 or hMPV/NL/1/99. The animal sera were serially twofold diluted and incubated with 100 p.f.u. hMPV in the presence of guinea pig complement for 1 h at 4 °C. The virus/serum mixtures were transferred to Vero cell monolayers and overlaid with OPTI-MEM containing 1 % methylcellulose. After 6 days of incubation at 37 °C, the monolayers were immunostained using hMPV ferret polyclonal antiserum for quantification. Neutralization titres were expressed as the reciprocal log2 of the highest serum dilution that inhibited 50 % of virus plaques.
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RESULTS |
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Identification and characterization of small-animal models supporting hMPV replication in the respiratory tract
To identify a small-animal model that will support hMPV replication in the respiratory tract, hamsters, mice, cotton rats and ferrets, previously used to study other paramyxoviruses, were investigated. Small animals have been employed as models to screen attenuation phenotypes of live virus vaccines as well as to evaluate the immunogenicity elicited by the virus vaccine candidates. Syrian golden hamsters have been used to characterize live attenuated PIV3 vaccine candidates (Haller et al., 2000; Skiadopoulos et al., 1999
). Live attenuated candidate RSV vaccines have been studied in cotton rats and BALB/c mice (Jin et al., 2000
). Ferrets have been employed to study attenuation phenotypes of live influenza virus vaccines (Maassab et al., 1982
). Therefore, these animals were chosen initially to study their permissiveness to hMPV infection. The animals were dosed intranasally with 106 p.f.u. hMPV, and 4 days post-infection the nasal turbinate and lung tissues were assayed for virus replication. The results showed that Syrian golden hamsters and ferrets supported hMPV replication in the respiratory tract to high titres. As shown in Table 1
, hMPV titres of 5·3 and 4·3 log10 p.f.u. (g tissue)1 in the URT and LRT, respectively, of hamsters were observed. Ferret nasal and lung tissues yielded hMPV titres of 4·7 and 4·0 log10 p.f.u. (g tissue)1, respectively. The body temperature of the ferrets was monitored daily but the animals did not develop a fever during the course of virus infection. Signs of illness such as a cold, runny nose, sneezing and loss of appetite were not observed for either hamsters or ferrets. Both hamsters and ferrets developed neutralizing hMPV antibodies ranging in titre from 3 to 8 reciprocal log2 in the individual animals 4 weeks post-infection (data not shown). In contrast, BALB/c mice displayed hMPV replication titres of only 3·4 and 2·4 log10 p.f.u. (g tissue)1 in the URT and LRT, respectively (Table 1
). hMPV replication was not observed in either nasal turbinate or lung tissue of infected cotton rats (Table 1
), which is in contrast to RSV. To study further the kinetics of hMPV replication in vivo, a time course was carried out in hamsters for 6 days (Table 2
). Days 3 and 4 post-infection displayed the highest levels of hMPV replication. hMPV titres of 4·55·7 log10 p.f.u. (g tissue)1 in the URT and 4·34·8 log10 p.f.u. (g tissue)1 in the LRT were observed. The hamsters appeared to clear the virus infection by day 6. Hamsters are more cost-effective than ferrets and immunological reagents for hamsters are more readily available than for ferrets. Therefore, Syrian golden hamsters were chosen as a small-animal model to analyse further the immune response as well as replication of hMPV from other subgroups.
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To study whether immunization with hMPV from subgroup A can protect from subsequent subgroup B infection, cross-challenge studies were carried out in hamsters (Table 3). The animals received 105 p.f.u. hMPV/NL/1/00 or hMPV/NL/1/99 intranasally. Four days later, six animals were sacrificed and virus titres in the nasal turbinate and lung tissue were determined. Hamsters that received hMPV/NL/1/00 displayed titres of 5·7 and 4·8 log10 p.f.u. (g tissue)1 in the URT and LRT, respectively. The animals that were infected with hMPV/NL/1/99 replicated to titres of 4·2 and 5·0 log10 p.f.u. (g tissue)1 in the URT and LRT, respectively (Table 3
). Separate groups of hamsters immunized with hMPV/NL/1/00 or hMPV/NL/1/99 were challenged on day 28 post-infection with both hMPV/NL/1/00 and hMPV/NL/1/99. The results showed that animals vaccinated with hMPV/NL/1/00 were completely protected from both hMPV subgroups A and B. Similarly, hamsters that received hMPV/NL/1/99 were completely protected from challenge with hMPV/NL/1/00 or hMPV/NL/1/99 (Table 3
). Only the animals that were administered placebo medium showed high levels of
5 log10 p.f.u. (g tissue1) of hMPV subgroup A or B replication in the URT and LRT (Table 3
). This result demonstrated that both hMPV subgroups A and B have the ability to replicate to high titres in the respiratory tract of hamsters if the dose is >105 p.f.u. In hamsters, infection with subgroup A hMPV produced an immune response that conferred protection from subsequent subgroup B infection. Similarly, hMPV subgroup B-immunized hamsters were protected from subgroup A challenge.
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Briefly, hMPV-seronegative rhesus monkeys were infected intratracheally and intranasally with a 1 ml dose at each site containing 1·3x105 p.f.u. hMPV. Virus shedding was monitored daily for 11 days post-infection in the nasopharynx and on days 1, 3, 5, 7 and 9 post-infection in the trachea. From the four rhesus monkeys that received hMPV/NL/1/00, only a single animal showed a virus titre of 4·0 log10 p.f.u. ml1 in the URT (Table 5). This animal shed virus for 5 days in the URT and for only 3 days in the LRT where hMPV titres of 1·8 log10 p.f.u. ml1 were observed. The other three rhesus monkeys displayed very low levels of virus shedding of 1·31·8 log10 p.f.u. ml1 in the URT and titres ranging from 1·3 log10 p.f.u. ml1 to below the assay detection limit in the LRT. The mean hMPV peak replication titre in the nasopharynx of rhesus monkeys was 2·2 log10 p.f.u. ml1 and 1·3 log10 p.f.u. ml1 in the trachea (Table 5
). Neutralizing serum antibody titres were not determined, since only low levels of replication were observed.
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The kinetics of hMPV replication in the URT and LRT during the course of infection was compared with that observed for RSV (Fig. 1). The progress of infection for two animals each infected with hMPV or RSV in the URT showed that virus shedding lasted for >7 days post-infection. The peak of virus replication in the URT occurred between days 4 and 5 for hMPV and days 6 and 8 for RSV A2 (Fig. 1A
) Virus shedding in the LRT showed that hMPV reached peak titres between 5 and 7 days post-infection and RSV displayed peak titres on day 5 post-infection with similar time periods of virus shedding (Fig. 1B
).
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DISCUSSION |
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Therefore, a number of small-animal and primate models were evaluated for permissiveness to hMPV infection. Animal models currently used to evaluate RSV or hPIV3 vaccines or mAbs support high levels of RSV or PIV3 replication in the respiratory tract. Usually virus titres of 104105 p.f.u. ml1 are observed in the URT and LRT of RSV or PIV3-immunized animals (Haller et al., 2000; Jin et al., 2000
, 2003
; Pennathur et al., 2003
). High levels of virus replication in the animal models are desirable for the following reasons: (i) The degree of attenuation of vaccine strains can be better measured. Reduced replication in the respiratory tract of animals should correlate with reduced disease in humans. (ii) High levels of virus replication in the animals should stimulate strong cellular and humoral immune responses and elicit high neutralizing antibody titres, in general a correlate of immune protection against respiratory virus infections. (iii) Immune protection of vaccinated animals will result in a marked reduction in challenge virus titres (>2 log10) demonstrating the effectiveness of the vaccine.
Four small-animal models, mice, hamsters, cotton rats and ferrets, were tested for permissiveness to hMPV infection, replication in the respiratory tract and induction of neutralizing antibodies. The results showed that Syrian golden hamsters and ferrets supported hMPV replication to high titres of 45 log10 p.f.u. ml1 in the URT and LRT. None of the animals showed signs of hMPV disease. Hamsters appeared to clear most of the hMPV infection by day 6 post-infection. Hamsters and ferrets produced a neutralizing antibody response, displaying titres ranging from 3 to 8 log2 for the individual animals. BALB/c mice were semi-permissive for hMPV infection and cotton rats did not display detectable hMPV titres in the nasal turbinates or lungs 4 days post-infection.
Two primate models, rhesus macaques and AGMs, were evaluated as potential hMPV primate models. Rhesus monkeys were not very permissive for hMPV infection and did not display high hMPV titres in the URT and LRT, even though a dose of >105 p.f.u. was administered intranasally and intratracheally. Only one of four animals displayed an hMPV titre of 4 log10 p.f.u. ml1 in the URT; the other three animals shed only low levels of hMPV. hMPV replication in the LRT of rhesus monkeys was not detected in two animals and was very low (1·3 and 1·8 log10 p.f.u. ml1) in the other two animals. In contrast, hMPV mean replication titres of 3·7 and 5·0 log10 p.f.u. ml1 were observed in the nasopharynx and trachea of AGMs, respectively. The results obtained for hMPV replication in rhesus monkeys and AGMs were similar to those observed for RSV. It has previously been demonstrated that cynomolgus macaques are only semi-permissive for RSV infection, while AGMs support high levels of RSV replication (A. Haller, unpublished observation; Jin et al., 2003). In AGM sera collected 28 days post-hMPV/NL/1/00 infection, mean hMPV neutralizing antibody titres of 9 log2 were observed for the homologous antigen. Slightly lower neutralizing antibody levels were observed when the heterologous virus (hMPV/NL/1/99) was used in the neutralization assay. Similar levels of RSV neutralizing antibody titres have been observed for sera obtained from RSV-infected AGMs (Jin et al., 2003
). It is expected that AGMs will also be permissive for hMPV subgroup B infection. Neither rhesus monkeys nor AGMs displayed signs of hMPV disease. Therefore, AGMs did not mimic the hMPV infection observed in humans. The observation that sera obtained from hMPV-infected AGMs contained similar levels of subgroup A and B neutralizing antibody titres suggested that the immune systems of non-human primates and hamsters are different in nature. Hamsters appeared to produce more hMPV subgroup-specific neutralizing antibody classes, while AGMs elicited antibodies that neutralized both subgroups equally well.
Syrian golden hamsters were used to study whether infection with hMPV subgroups A or B could generate an immune response that would cross-protect. These findings have implications on vaccine design and the number of vaccine strains necessary for immune protection from wild-type hMPV infection. The results showed that hamsters vaccinated with hMPV/NL/1/00 (subgroup A) were protected from challenge with hMPV/NL/1/99, a subgroup B representative. Similarly, hamsters that had received a subgroup B virus (hMPV/NL/1/99) were protected from challenge with a subgroup A virus (hMPV/NL/1/00). These results suggest that an hMPV vaccine based on either the subgroup A or subgroup B genetic backbone can provide sufficient protection from infection by other hMPV subgroups. This is an important finding that will facilitate rational vaccine design, since future hMPV vaccines may not need to contain both hMPV subgroups to be efficacious. hMPV subgroup cross-protection ability was likely due to the highly conserved hMPV F protein, although some contributions may stem from the less conserved hMPV G protein. The hMPV F protein is a viral surface glycoprotein and is thought to be responsible in part for eliciting the neutralizing antibody response. Although the hMPV neutralizing antibody titres for the heterologous hMPV antigen were lower by as much as 4 log2, the neutralizing antibodies induced effectively neutralized and protected the hamsters from the heterologous hMPV challenge virus. Based on the importance of cell-mediated immunity for humans to recover from paramyxovirus infections, it is likely that T cell responses to hMPV also contributed to the high level of heterologous protection observed after hMPV challenge.
Similar observations have been made for RSV, a related paramyxovirus. For RSV, subgroup A can provide immunological protection from subgroup B (Jin et al., 2003). Therefore, an RSV vaccine based on subgroup A should be sufficient to protect from RSV subgroup B. The RSV F proteins of subgroups A and B are highly conserved and display an 89 % amino acid identity, while the G proteins are divergent and only show 53 % identity. In contrast, hPIV3 will not induce an immune response that will protect from hPIV1 infection (Tao et al., 2000
). The lack of immune cross-protection is most likely due to a greater degree of divergence of the surface glycoproteins of hPIV3 and hPIV1. The F and haemagglutininneuraminidase (HN) proteins of hPIV3 and hPIV1 display amino acid identities of only 41·9 and 35·5 %, respectively. These results suggest that immune cross-protection requires at least one highly conserved viral surface glycoprotein among the different virus strains to elicit cross-reactive neutralizing antibodies. The high degree of hMPV F protein conservation suggests that antibodies directed against the hMPV F protein should neutralize both subgroups A and B of hMPV, which is supported by the results obtained from hamsters presented in this study.
In summary, this study identified two small-animal models, hamsters and ferrets, that supported efficient hMPV replication in the respiratory tract and produced high hMPV neutralizing antibody titres. The hamster studies showed that hMPV subgroups A and B could elicit cross-subgroup immune protection and this finding will influence rational vaccine design. AGMs were also shown to be permissive for hMPV infection, and high levels of hMPV replication were observed in the respiratory tract. Furthermore, hMPV infection of AGMs induced a high level of hMPV neutralizing antibodies. These results will have an important impact on future hMPV vaccine design and facilitate evaluation of hMPV vaccines, mAbs and antivirals.
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ACKNOWLEDGEMENTS |
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Received 13 November 2003;
accepted 13 February 2004.