| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Department of Virology, Erasmus MC, Rotterdam, The Netherlands
2 MedImmune, Inc., 1 MedImmune Way, Gaithersburg, MD 20878, USA
Correspondence
Bernadette G. van den Hoogen
b.vandenhoogen{at}erasmusmc.nl
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Clinical manifestations of hMPV infections are similar to those caused by respiratory syncytial virus (RSV), ranging from mild respiratory illness to bronchiolitis and pneumonia (van den Hoogen et al., 2003; Williams et al., 2006). RSV and hMPV are both members of the subfamily Pneumovirinae within the family Paramyxoviridae. However, within this subfamily, RSV is a member of the genus Pneumovirus and hMPV is the first mammalian member of the genus Metapneumovirus, based on differences in genome organization (van den Hoogen et al., 2002). Phylogenetic analysis of fusion (F) and attachment (G) genes of a large number of hMPV isolates revealed the existence of two main genetic virus lineages (A and B), each divided into at least two sublineages (1 and 2). The two main lineages, with prototype viruses NL/1/00 and NL/1/99 for lineages A and B, respectively, were found to be antigenically distinct in virus-neutralization assays with ferret sera (van den Hoogen et al., 2004).
The genome of HMPV encodes three surface glycoproteins: F, G and the small hydrophobic protein (SH). Whereas the G and SH proteins are highly variable, the F protein is highly conserved between the major lineages A and B. Antibodies induced against the F protein are correlates of protection in animal models (Skiadopoulos et al., 2006; Tang et al., 2005). The fusion protein of hMPV demonstrates similarities to those of other paramyxoviruses (van den Hoogen et al., 2002, 2004). The F proteins of paramyxoviruses are class I viral F proteins, which are synthesized as an inactive precursor F0 that must be cleaved by host endoproteases, resulting in two disulfide-linked polypeptides, F1 and F2 (Lamb et al., 2006; Russell et al., 2001).
The clinical impact of hMPV warrants the development of vaccines. For RSV, subunit vaccines based on the F or G proteins have been developed and tested in a variety of rodent and primate models, where they demonstrated protective efficacy (Hancock et al., 2000; Murphy et al., 1989; Walsh et al., 1987). In addition, RSV F subunit vaccines have been evaluated in clinical trials in healthy adults, children older than 1 year with or without underlying pulmonary disease, the elderly, and pregnant women, and have been found to be safe and effective (Falsey & Walsh, 1996; Munoz et al., 2003; Piedra et al., 2003; Tristram et al., 1993). Based on the genetic relationship of RSV and hMPV, we hypothesized that a similar subunit vaccine for hMPV would induce protective immunity against hMPV infection. Soluble F proteins of prototype viruses representing the two main lineages of hMPV were produced to high yields in stable NSO murine myeloma cell lines (Ulbrandt et al., 2006). We evaluated the antigenicity, immunogenicity and cross-protective efficacy of these soluble hMPV F proteins in Syrian golden hamsters (Mesocricetus auratus). As adjuvants are known to enhance the immune response, the immunogenicity of the soluble F proteins was tested in combination with different adjuvants: Alum, Specol and iscom matrix (Stills, 2005). Alum induces low antibody titres when used with subunit vaccines, but it was chosen because of its long history of use in humans. Iscom matrix is known to induce high antibody titres and is currently being evaluated in humans. Specol was chosen to function as positive control, as it induces high antibody titres in rodents.
This study demonstrates that soluble hMPV F proteins adjuvanted with Specol or iscom matrix are promising candidate subunit vaccines for the induction of protective antibody levels against homologous and heterologous hMPV infections.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
) were grown in Iscove's modified Dulbecco's medium (IMDM; BioWhittaker) supplemented with 10 % fetal calf serum (Greiner Bio-One), 100 IU penicillin ml–1, 100 µg streptomycin ml–1 and 2 mM glutamine. The construction of recombinant hMPV NL/1/00 (prototype virus for lineage A) and NL/1/99 (prototype virus for lineage B) has been described previously (Herfst et al., 2004). High-titre virus stocks were produced in Vero-118 cells in infection medium: IMDM supplemented with 4 % BSA fraction V (Invitrogen), 100 IU penicillin ml–1, 100 µg streptomycin ml–1, 2 mM glutamine and 3.75 µg trypsin ml–1. After 7 days, the cultures were harvested and stored in 25 % sucrose at –70 °C.
Construction of soluble F proteins.
Constructs expressing a truncated version of the hMPV F protein, lacking the transmembrane domain, were generated as described previously (Ulbrandt et al., 2006). In brief, full-length hMPV cDNA plasmids were used as templates to generate F gene PCR products by using the following oligonucleotides for both the NL/1/00 and NL/1/99 sequences: 5'-AATCAACGGTCCGCCACCATGTCTTGGAAAGTG-3' and 5'-TTAATTGAATTCTTAGTGATGGTGATGGTGATGGCCAGTGTTTCCTTTCTCTGC-3'. The PCR products were digested with restriction endonucleases RsrII and EcoRI and ligated to the pEE15.1 vector (Lonza) digested with the same restriction endonucleases. Stable NSO murine myeloma cell lines expressing NL/1/00 or NL/1/99 F proteins were generated as described by Bebbington et al. (1992). Soluble F protein (Fsol) was purified by affinity chromatography using an F-specific mAb, 1017, which binds to both NL/1/00 and NL/1/99 F proteins (Ulbrandt et al., 2006), coupled to cyanogen bromide-activated Sepharose and eluted with 0.1 M glycine, pH 2.8. The eluate was neutralized with 0.1 vol. 1 M Tris/HCl, pH 8.0, and was dialysed against PBS.
Vaccine preparations.
For the iscom matrix-adjuvanted Fsol preparation, N-decanoyl-N-methylglucamine (MEGA-10; Sigma-Aldrich) at a final concentration of 2 % was supplemented with cholesterol [1 mg (mg protein)–1; Sigma], phosphatidylethanolamine [1 mg (mg protein)–1; Sigma] and Quil-laja glucosides [5 mg ISCOPREP 703 (mg protein)–1; Isotec) and dialysed against PBS. The iscom matrix preparation was analysed by negative-contrast electron microscopy, revealing the typical iscom matrix particles with a diameter of approximately 40 nm. The iscom matrix preparation was added to the F protein immediately before immunization, resulting in preparations containing 10 µg Fsol in 100 µl.
Concerning the correct choice of an aluminium-containing adjuvant, the charge of adjuvants and antigens is characterized by the point of zero charge (PZC) and isoelectric point (pI), respectively (al-Shakhshir et al., 1994). Maximum adsorption of protein by aluminium adjuvants is reached at pH conditions under which the antigen and adjuvant have opposite charges. Based on the approximate pI of 5.9 of the Fsol proteins (http://au.expasy.org/cgi-bin/pi-tool), Alhydrogel with PZC value 11.1 was chosen as aluminium-containing adjuvant (a gift from Dr Erik B. Lindblad, Brenntag Biosector, Frederikssund, Denmark). A vaccine dose was prepared by mixing 50 µl Fsol (200 µg protein ml–1 in PBS) with 50 µl 2 % Alhydrogel (10.3 mg Al3+ ml–1), resulting in preparations containing 10 µg Fsol and 0.52 mg Al3+.
Doses of Specol vaccine (Stimune; Cedi Diagnostics) were prepared by mixing 4 vols water phase containing 10 µg Fsol with 5 vols Specol while mixing vigorously.
To obtain inactivated whole-virus vaccines, hMPV strains NL/1/00 and NL/1/99 were grown to 70–90 % cytopathic effect on Vero-118 cells. After one freeze–thaw cycle, cell-free supernatants were purified and concentrated by using a 30–60 % (w/w) sucrose gradient. Virus was inactivated with 
-propiolactone (Sigma-Aldrich) and complete inactivation was confirmed by titration on Vero-118 cells. SDS-PAGE analysis and Western blotting with an F monoclonal antibody (mAb) indicated that 100 µg inactivated virus contained approximately 10 µg F protein (data not shown); therefore, a vaccine dose of 100 µg inactivated virus (in 100 µl PBS) was used.
Animal experiments.
All intramuscular immunizations, intranasal inoculations, orbital punctures and euthanasia were performed under anaesthesia with inhaled isoflurane. All animal studies were approved by an independent, national animal ethics committee (DEC Consult) and the Dutch authority for working with genetically modified organisms, and were carried out in accordance with animal-experimentation guidelines.
Immunization and challenge.
Five- to seven-week-old female Syrian golden hamsters (Harlan Sprague–Dawley) were immunized twice intramuscularly at a 3 week interval with a dose of 100 µl vaccine containing 10 µg Fsol or 100 µg inactivated virus in PBS. Three weeks after the second immunization in the immunization-optimization experiment, or 4 days post-infection in the challenge experiment, blood samples were collected by orbital puncture. Blood samples were stored overnight at room temperature and centrifuged for 15 min at 1200 g; serum was collected and stored at –20 °C.
Animals were challenged intranasally with 100 µl NL/1/00 virus, diluted in PBS in order to obtain the required dose [104, 105 or 106 TCID50 (50 % tissue culture infectious dose) in 100 µl]. Four days after inoculation, lungs and nasal turbinates were collected, snap-frozen immediately and stored at –80 °C until further processing.
F protein-specific ELISA.
Ninety-six-well plates were coated overnight at 4 °C with 100 ng Fsol protein (NL/1/00 and NL/1/99, ratio 1 : 1) per well in PBS. Serum samples, diluted in Meddens reagent (Meddens), were added to the plates and incubated for 1 h at 37 °C. After washing, plates were incubated for 1 h with goat anti-hamster IgG–horseradish peroxidase (1 : 5000; DakoCytomation). 3,3',5,5'-Tetramethylbenzidine (TMB) diluted 1 : 10 in TMB diluent (Meddens) was used as a substrate. The reaction was stopped by adding an equal volume of 2 M H2SO4, after which the A450 was determined. Results are depicted after subtraction of background values.
Plaque-reduction virus neutralization (PRVN) assay.
Virus-neutralizing (VN) antibody titres were determined by a PRVN assay. Heat-inactivated (30 min, 56 °C) serum samples, diluted by 2–3, 2–5, 2–7 and 2–9, were incubated for 1 h at 37 °C with 50 p.f.u. NL/1/00 or NL/1/99 expressing the enhanced green fluorescent protein (EGFP) at position 3 of the genome. Subsequently, the serum–virus mixtures were added to Vero-118 cells in 24-well plates and incubated at 37 °C on a rocking platform. After 2 h, the supernatants were removed and 1 ml IMDM containing 2 % BSA fraction V, 1 % methylcellulose (MSD) and 3.75 µg trypsin ml–1 was added to the wells. Six days later, fluorescent plaques were counted by using a Typhoon 9410 variable-mode imager (GE Healthcare). VN antibody titres are expressed as the dilution resulting in 50 % reduction of the number of plaques, calculated according to the method of Reed & Muench (1938). Per assay, each serum was tested in duplicate against hMPV NL/1/00 and NL/1/99.
Virus titrations.
Tissues from the inoculated hamsters were homogenized by using a Polytron homogenizer (Kinematica AG) in infection medium. After removal of tissue debris by centrifugation, supernatants were used for virus titration in Vero-118 cells. Titrations were performed with tenfold serial dilutions in 96-well plates (Greiner Bio-One). Confluent monolayers of Vero-118 cells were spin-inoculated (15 min, 2000 g) with 100 µl tenfold serial dilutions of each sample and incubated at 37 °C. Two hours after the spin inoculation, the inoculum was replaced with fresh infection medium. After 3–4 days, 100 µl fresh infection medium was added to each well. Seven days after inoculation, infected wells were identified by immunofluorescence assays with hMPV-specific polyclonal antiserum raised in guinea pigs, as described previously (van den Hoogen et al., 2001). Titres, expressed as TCID50, were calculated as described by Reed & Muench (1938). Titres were calculated (g tissue)–1, with a detection limit of 101.6 and 101.2 TCID50 (g tissue)–1 for nasal turbinates and lungs, respectively.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
), except for viral shedding from the nose at 4 days p.i. at the lowest inoculation dose (Fig. 1a). At 2 and 4 days p.i., animals inoculated with 106 TCID50 harboured more virus in their nose and lungs than did those inoculated with lower virus doses. Only at 4 days p.i., and only with the two highest doses (105 and 106), was virus detected in the lungs of all animals (Fig. 1b). Based on these results, challenge experiments were conducted with 106 TCID50 with subsequent euthanasia at 4 days p.i.
|
. By using these stable NSO murine myeloma cell lines, we were able to affinity-purify up to 20 mg soluble hMPV F protein ml–1. Analyses of these proteins by SDS-PAGE and Coomassie blue staining demonstrated that the affinity-purified proteins were >90 % pure, with a predicted molecular mass of around 55 kDa (Fig. 2).
|
Six Syrian golden hamsters per group were immunized twice intramuscularly with 10 µg Fsol alone, Fsol with adjuvant (iscom matrix, alum or Specol), 100 µg inactivated virus or PBS. Three weeks later, sera were subjected to F-specific ELISA at a 1 : 1600 dilution. All animals, except the PBS-immunized animals, demonstrated the presence of F-specific antibodies (Fig. 3). Animals immunized with an NL/1/00 F protein preparation displayed higher ELISA antibody titres than those immunized with NL/1/99 preparations, although a mixture of antigens from both prototype viruses was used to coat the ELISA plates. The non-adjuvanted Fsol proteins induced slightly lower antibody titres compared with the whole virus preparation. Addition of adjuvants increased the immunogenicity of the Fsol proteins, with the highest titres induced by iscom matrix- and Specol-adjuvanted vaccines (Fig. 3). High ELISA antibody titres correlated with high VN antibody titres (data not shown). Based on the high antibody titres induced by immunization, the protective efficacy of the iscom matrix- and Specol-adjuvanted F subunit vaccines was tested in immunization/challenge experiments.
|
Immunization of control animals with PBS, iscom matrix or Specol (without antigen) did not induce detectable virus-specific antibodies (Fig. 4), which upon challenge resulted in virus shedding in the nose of all animals (Fig. 5a). Unfortunately, in contrast to the animals in the challenge-optimization experiment (Fig. 1
), some of these control animals (e.g. three of eight, two of six and one of six for the PBS, iscom matrix and Specol groups, respectively) did not secrete virus from the lower respiratory tract (LRT) (Fig. 5b). Similarly, immunization of hamsters with Fsol alone induced low to undetectable levels of antibodies (Fig. 4). Upon challenge, virus was detected in the upper respiratory tracts (URTs) of all these animals and in the LRTs of five and six of eight animals (Fsol/1/99 and Fsol/1/00, respectively) (Fig. 5).
|
|
). Upon challenge, these antibody titres partly protected from infection of the URT, with viral titres in the nose being statistically significantly lower than those observed in the nasal turbinates of the PBS-immunized animals (Mann–Whitney test, P<0.05). Complete (cross-) protection of the LRT was induced by immunization with these adjuvanted Fsol proteins. None of the animals demonstrated the presence of infectious virus (Fig. 5b) or viral genomes in their lung samples (data not shown). Immunization with adjuvanted Fsol vaccines induced significantly higher protective efficacy for LRT than did immunization with PBS (Mann–Whitney test, P=0.038). Immunization with adjuvanted Fsol/1/00 as well as Fsol/1/99 (cross-) protected the LRT against challenge infection with hMPV/NL/1/00.
All sera were tested in PRVN assays against NL/1/00 (Fig. 4a) and NL/1/99 (Fig. 4b). Sera from all animals from the control groups (PBS, iscom matrix or Specol) were negative for neutralizing antibodies. Animals immunized with adjuvanted vaccines had higher antibody titres than those immunized with Fsol alone. In general, homologous VN antibody titres were higher than heterologous VN antibody titres, with the highest homologous titres observed in the NL/1/00-immunized animals. Mean homologous titres induced by the adjuvanted vaccines were 20–30-fold and 2.5–3.4-fold higher than the heterologous titres for NL/1/99 and NL/1/00, respectively (Table 1). The difference in homologous and heterologous titres was not reflected in differences in protective efficacy, as both homologously and heterologously challenged animals were protected from LRT infection and displayed an equal reduction of viral titres in nasal turbinates.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Munoz et al., 2003; Piedra et al., 2003; Tristram et al., 1993). We have produced recombinant soluble F proteins (Fsol proteins) for the prototypes of the two main lineages of hMPV (NL/1/00, lineage A; NL/1/99, lineage B) in a system that allows production of high yields of recombinant proteins. The use of these proteins as subunit vaccines was evaluated in Syrian golden hamsters.
The F proteins of paramyxoviruses are class I viral F proteins, which are synthesized as an inactive precursor, F0. This immature F protein is subsequently modified by the addition of N-linked carbohydrate and then assembled into homo-oligomers in the rough endoplasmic reticulum. F0 is subsequently cleaved endoproteolytically, resulting in two polypeptides, F1 and F2, that are linked covalently to each other by disulfide bonds (Lamb et al., 2006; Russell et al., 2001). Thus, the F protein is present during infection in immature and mature forms (Sakurai et al., 1999).
The F glycoprotein is one of the major targets of RSV- or hMPV-neutralizing antibodies (Skiadopoulos et al., 2006; Tang et al., 2005) and is therefore an ideal candidate to use as subunit vaccine. Upon vaccination and/or natural infection with RSV, both neutralizing and non-neutralizing antibodies can be detected (Tsui et al., 1996). Several studies have suggested that non-neutralizing antibodies are directed against the uncleaved form of the F protein, whilst neutralizing antibodies are directed against the cleaved form of the protein (Sakurai et al., 1999; Tsui et al., 1996).
Both Fsol proteins used in this study were produced from viruses containing a serine at position 101 in the RQSR motif at the putative cleavage site of the F protein. It has previously been demonstrated that hMPV expressing an F protein with 101S is incapable of initiating multicycle growth without the addition of trypsin, as the F protein was not efficiently cleaved proteolytically (Schickli et al., 2005). In the present study, the production of the Fsol proteins of hMPV occurred in the presence of serum and in the absence of trypsin, so most of the Fsol proteins were not cleaved proteolytically. This was also confirmed by performing Western blot analysis on the Fsol proteins after incubation with or without trypsin. After the addition of trypsin, a smaller band was revealed, corresponding to the predicted size of cleavage fragment hMPV F1, by using F-specific mAb 1017 (data not shown).
Although most of the proteins used for the vaccine preparation were uncleaved, we have demonstrated that these truncated Fsol proteins were as immunogenic as the proteins in the native form. Immunization with the soluble proteins induced neutralizing-antibody titres similar to those induced by immunization with whole inactivated virus. However, antibody titres in animals immunized with Fsol were low. Therefore, several adjuvants were tested for their potential to induce higher neutralizing-antibody titres.
Alum was chosen because of its long history of use in humans, although it is known to induce low antibody titres when used with subunit vaccines (Gupta, 1998). Specol and iscom matrix were chosen because they both generate long-lasting, functional antibody responses, but neither adjuvant is licensed for human use (Beck et al., 2003; Leenaars et al., 1994; Sanders et al., 2005). However, several studies in human volunteers have shown that iscom-based vaccines are highly immunogenic, as well as safe and well-tolerated (Davis et al., 2004; Frazer et al., 2004). The addition of Alum to the Fsol proteins resulted in antibody levels similar to those induced by the non-adjuvanted Fsol. This weak response might be the result of a low degree of adsorption of Fsol to Alhydrogel, which is considered to be an important parameter for the function of aluminium adjuvants (Lindblad, 2004).
Addition of both Specol and iscom matrix to the Fsol protein enhanced the hMPV-specific antibody response. This enhanced effect was also seen in cotton rats immunized with 25 µg soluble hMPV F protein adjuvanted with Titermax Gold (Cseke et al., 2007). Although Titermax Gold is known to induce high antibody titres, the adjuvant induced sterile abscesses in non-human primates and is not licensed for human use (Deng et al., 2002).
The iscom matrix- and Specol-adjuvanted F-subunit vaccines were tested for protective efficacy against homologous and heterologous infection in Syrian golden hamsters. For this purpose, we developed a challenge model for NL/1/00 infections in Syrian golden hamsters. An optimum inoculation dose of 106 TCID50, followed by collection of lungs and nasal turbinates at 4 days p.i., resulted in virus detection in samples collected from all animals.
Using this challenge model in an immunization/challenge experiment, none of the vaccines induced complete protection against URT infection, although viral titres in the nose of adjuvanted Fsol-immunized animals were significantly lower than those in the PBS-immunized animals. However, the primary goal of immunization against respiratory viruses is prevention of serious LRT illnesses. The control vaccines (iscom matrix or Specol without antigen, PBS or non-adjuvanted Fsol) did not prevent LRT infections, whilst immunization with adjuvanted Fsol induced complete protection against LRT infection. Even after optimization of the challenge model, subsequent challenges of the control animals in the immunization/challenge experiment resulted in <100 % infection. Although intranasal inoculation of Syrian golden hamsters has been used successfully as the infection procedure in other studies, this route of infection might be less efficient for the virus strains used in the present study. Perhaps a higher inoculum dose or even intratracheal infection would have resulted in 100 % infection. However, intranasal infection, with a risk for a less robust infection, resembles a natural infection more closely. Despite the fact that not all control animals harboured virus in their lungs upon challenge, significant protective efficacy was demonstrated for the adjuvanted Fsol proteins. All animals immunized with Fsol adjuvanted with iscom matrix or Specol were protected from infection of their lungs, compared with three of eight animals in the PBS-immunized group (Mann–Whitney test, P=0.038).
In passive-transfer experiments in which RSV-specific mAbs were used, it was demonstrated that URT protection requires significant higher antibody doses than does protection of the LRT (Siber et al., 1994). This suggests that antibody titres induced by the studied vaccines might be too low to protect against URT infection. A third vaccination could be considered to increase the antibody levels or the use of alternative adjuvants in order to achieve protection of the URT. Alternatively, although the uncleaved Fsol proteins induced neutralizing antibodies, immunization with a cleaved form of Fsol might induce higher neutralizing-antibody titres that protect against URT infection. Both the adjuvanted Fsol/1/00 and Fsol/1/99 proteins induced higher homologous than heterologous VN antibody titres, indicating serological differences between the two main lineages of hMPV, as described previously (van den Hoogen et al., 2004). However, antibodies raised against the NL/1/99 F protein provided cross-protection against heterologous NL/1/00 virus infection of the LRT. Although we demonstrate cross-protection in only one direction, it seems likely that cross-protection will be obtained in both directions. This has been observed previously with the F protein in its native form (Skiadopoulos et al., 2004; Tang et al., 2005).
Vaccine development aimed at protecting naïve individuals from infection with RSV or hMPV is hampered by an experience with a formalin-inactivated RSV vaccine. Immunization of naïve children with this vaccine induced enhanced disease upon subsequent infection (Kim et al., 1969). Enhanced disease has so far not been observed in employment of F subunit vaccines for RSV in patients with pre-existing immunity and is not to be expected for hMPV subunit vaccines. Therefore, the focus of this study was the protective efficacy of F subunit vaccines. Syrian golden hamsters are not the ideal animal model to study vaccine-induced immune pathology, as immunological tools are scarce. The presented results justify subsequent studies in non-human primates, in which the safety issue can be addressed properly.
Our results demonstrate that immunization of Syrian golden hamsters with adjuvanted hMPV Fsol subunit vaccines induced a strong antibody response that provided complete protection against LRT infection with homologous and heterologous viruses. The availability of this protein in high quantities, in combination with the demonstrated beneficial use of the safe adjuvant iscom matrix, makes this F subunit vaccine an excellent candidate for further exploration to boost antibody titres in humans with pre-existing antibody titres.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Bebbington, C. R., Renner, G., Thomson, S., King, D., Abrams, D. & Yarranton, G. T. (1992). High-level expression of a recombinant antibody from myeloma cells using a glutamine synthetase gene as an amplifiable selectable marker. Biotechnology (N Y) 10, 169–175.[CrossRef][Medline]
Beck, I., Gerlach, H., Burkhardt, E. & Kaleta, E. F. (2003). Investigation of several selected adjuvants regarding their efficacy and side effects for the production of a vaccine for parakeets to prevent a disease caused by a paramyxovirus type 3. Vaccine 21, 1006–1022.[CrossRef][Medline]
Cseke, G., Wright, D. W., Tollefson, S. J., Johnson, J. E., Crowe, J. E., Jr & Williams, J. V. (2007). Human metapneumovirus fusion protein vaccines that are immunogenic and protective in cotton rats. J Virol 81, 698–707.
Davis, I. D., Chen, W., Jackson, H., Parente, P., Shackleton, M., Hopkins, W., Chen, Q., Dimopoulos, N., Luke, T. & other authors (2004). Recombinant NY-ESO-1 protein with ISCOMATRIX adjuvant induces broad integrated antibody and CD4+ and CD8+ T cell responses in humans. Proc Natl Acad Sci U S A 101, 10697–10702.
Deng, X., Meyers, S. A., Tollner, T. L., Yudin, A. I., Primakoff, P. D., He, D. N. & Overstreet, J. W. (2002). Immunological response of female macaques to the PH-20 sperm protein following injection of recombinant proteins or synthesized peptides. J Reprod Immunol 54, 93–115.[CrossRef][Medline]
Falsey, A. R. & Walsh, E. E. (1996). Safety and immunogenicity of a respiratory syncytial virus subunit vaccine (PFP-2) in ambulatory adults over age 60. Vaccine 14, 1214–1218.[CrossRef][Medline]
Frazer, I. H., Quinn, M., Nicklin, J. L., Tan, J., Perrin, L. C., Ng, P., O'Connor, V. M., White, O., Wendt, N. & other authors (2004). Phase 1 study of HPV16-specific immunotherapy with E6E7 fusion protein and ISCOMATRIX adjuvant in women with cervical intraepithelial neoplasia. Vaccine 23, 172–181.[CrossRef][Medline]
Gupta, R. K. (1998). Aluminum compounds as vaccine adjuvants. Adv Drug Deliv Rev 32, 155–172.[CrossRef][Medline]
Hancock, G. E., Smith, J. D. & Heers, K. M. (2000). Serum neutralizing antibody titers of seropositive chimpanzees immunized with vaccines coformulated with natural fusion and attachment proteins of respiratory syncytial virus. J Infect Dis 181, 1768–1771.[CrossRef][Medline]
Herfst, S., de Graaf, M., Schickli, J. H., Tang, R. S., Kaur, J., Yang, C. F., Spaete, R. R., Haller, A. A., van den Hoogen, B. G. & other authors (2004). Recovery of human metapneumovirus genetic lineages a and B from cloned cDNA. J Virol 78, 8264–8270.
Kim, H. W., Canchola, J. G., Brandt, C. D., Pyles, G., Chanock, R. M., Jensen, K. & Parrott, R. H. (1969). Respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine. Am J Epidemiol 89, 422–434.
Kuiken, T., Van Den Hoogen, B. G., Van Riel, D. A., Laman, J. D., Van Amerongen, G., Sprong, L., Fouchier, R. A. & Osterhaus, A. D. (2004). Experimental human metapneumovirus infection of cynomolgus macaques (Macaca fascicularis) results in virus replication in ciliated epithelial cells and pneumocytes with associated lesions throughout the respiratory tract. Am J Pathol 164, 1893–1900.
Lamb, R. A., Paterson, R. G. & Jardetzky, T. S. (2006). Paramyxovirus membrane fusion: lessons from the F and HN atomic structures. Virology 344, 30–37.[CrossRef][Medline]
Leenaars, P. P., Hendriksen, C. F., Angulo, A. F., Koedam, M. A. & Claassen, E. (1994). Evaluation of several adjuvants as alternatives to the use of Freund's adjuvant in rabbits. Vet Immunol Immunopathol 40, 225–241.[CrossRef][Medline]
Lindblad, E. B. (2004). Aluminium compounds for use in vaccines. Immunol Cell Biol 82, 497–505.[CrossRef][Medline]
Munoz, F. M., Piedra, P. A. & Glezen, W. P. (2003). Safety and immunogenicity of respiratory syncytial virus purified fusion protein-2 vaccine in pregnant women. Vaccine 21, 3465–3467.[CrossRef][Medline]
Murphy, B. R., Sotnikov, A., Paradiso, P. R., Hildreth, S. W., Jenson, A. B., Baggs, R. B., Lawrence, L., Zubak, J. J., Chanock, R. M. & other authors (1989). Immunization of cotton rats with the fusion (F) and large (G) glycoproteins of respiratory syncytial virus (RSV) protects against RSV challenge without potentiating RSV disease. Vaccine 7, 533–540.[CrossRef][Medline]
Piedra, P. A., Cron, S. G., Jewell, A., Hamblett, N., McBride, R., Palacio, M. A., Ginsberg, R., Oermann, C. M. & Hiatt, P. W. (2003). Immunogenicity of a new purified fusion protein vaccine to respiratory syncytial virus: a multi-center trial in children with cystic fibrosis. Vaccine 21, 2448–2460.[CrossRef][Medline]
Reed, L. J. & Muench, H. (1938). A simple method of estimating fifty percent end points. J Hyg (Lond) 27, 493–497.
Russell, C. J., Jardetzky, T. S. & Lamb, R. A. (2001). Membrane fusion machines of paramyxoviruses: capture of intermediates of fusion. EMBO J 20, 4024–4034.[CrossRef][Medline]
Sakurai, H., Williamson, R. A., Crowe, J. E., Beeler, J. A., Poignard, P., Bastidas, R. B., Chanock, R. M. & Burton, D. R. (1999). Human antibody responses to mature and immature forms of viral envelope in respiratory syncytial virus infection: significance for subunit vaccines. J Virol 73, 2956–2962.
Sanders, M. T., Brown, L. E., Deliyannis, G. & Pearse, M. J. (2005). ISCOM-based vaccines: the second decade. Immunol Cell Biol 83, 119–128.[CrossRef][Medline]
Schickli, J. H., Kaur, J., Ulbrandt, N., Spaete, R. R. & Tang, R. S. (2005). An S101P substitution in the putative cleavage motif of the human metapneumovirus fusion protein is a major determinant for trypsin-independent growth in vero cells and does not alter tissue tropism in hamsters. J Virol 79, 10678–10689.
Siber, G. R., Leombruno, D., Leszczynski, J., McIver, J., Bodkin, D., Gonin, R., Thompson, C. M., Walsh, E. E., Piedra, P. A. & other authors (1994). Comparison of antibody concentrations and protective activity of respiratory syncytial virus immune globulin and conventional immune globulin. J Infect Dis 169, 1368–1373.[Medline]
Skiadopoulos, M. H., Biacchesi, S., Buchholz, U. J., Riggs, J. M., Surman, S. R., Amaro-Carambot, E., McAuliffe, J. M., Elkins, W. R., St Claire, M. & other authors (2004). The two major human metapneumovirus genetic lineages are highly related antigenically, and the fusion (F) protein is a major contributor to this antigenic relatedness. J Virol 78, 6927–6937.
Skiadopoulos, M. H., Biacchesi, S., Buchholz, U. J., Amaro-Carambot, E., Surman, S. R., Collins, P. L. & Murphy, B. R. (2006). Individual contributions of the human metapneumovirus F, G, and SH surface glycoproteins to the induction of neutralizing antibodies and protective immunity. Virology 345, 492–501.[CrossRef][Medline]
Stills, H. F., Jr (2005). Adjuvants and antibody production: dispelling the myths associated with Freund's complete and other adjuvants. ILAR J 46, 280–293.[Medline]
Tang, R. S., Mahmood, K., Macphail, M., Guzzetta, J. M., Haller, A. A., Liu, H., Kaur, J., Lawlor, H. A., Stillman, E. A. & other authors (2005). A host-range restricted parainfluenza virus type 3 (PIV3) expressing the human metapneumovirus (hMPV) fusion protein elicits protective immunity in African green monkeys. Vaccine 23, 1657–1667.[CrossRef][Medline]
Tristram, D. A., Welliver, R. C., Mohar, C. K., Hogerman, D. A., Hildreth, S. W. & Paradiso, P. (1993). Immunogenicity and safety of respiratory syncytial virus subunit vaccine in seropositive children 18–36 months old. J Infect Dis 167, 191–195.[Medline]
Tsui, P., Tornetta, M. A., Ames, R. S., Bankosky, B. C., Griego, S., Silverman, C., Porter, T., Moore, G. & Sweet, R. W. (1996). Isolation of a neutralizing human RSV antibody from a dominant, non-neutralizing immune repertoire by epitope-blocked panning. J Immunol 157, 772–780.[Abstract]
Ulbrandt, N. D., Ji, H., Patel, N. K., Riggs, J. M., Brewah, Y. A., Ready, S., Donacki, N. E., Folliot, K., Barnes, A. S. & other authors (2006). Isolation and characterization of monoclonal antibodies which neutralize human metapneumovirus in vitro and in vivo. J Virol 80, 7799–7806.
van den Hoogen, B. G., de Jong, J. C., Groen, J., Kuiken, T., de Groot, R., Fouchier, R. A. & Osterhaus, A. D. (2001). A newly discovered human pneumovirus isolated from young children with respiratory tract disease. Nat Med 7, 719–724.[CrossRef][Medline]
van den Hoogen, B. G., Bestebroer, T. M., Osterhaus, A. D. & Fouchier, R. A. (2002). Analysis of the genomic sequence of a human metapneumovirus. Virology 295, 119–132.[CrossRef][Medline]
van den Hoogen, B. G., van Doornum, G. J., Fockens, J. C., Cornelissen, J. J., Beyer, W. E., de Groot, R., Osterhaus, A. D. & Fouchier, R. A. (2003). Prevalence and clinical symptoms of human metapneumovirus infection in hospitalized patients. J Infect Dis 188, 1571–1577.[CrossRef][Medline]
van den Hoogen, B. G., Herfst, S., Sprong, L., Cane, P. A., Forleo, E., de Swart, R. L., Osterhaus, A. D. & Fouchier, R. A. (2004). Antigenic and genetic variability of human metapneumoviruses. Emerg Infect Dis 10, 658–666.[Medline]
Walsh, E. E., Hall, C. B., Briselli, M., Brandriss, M. W. & Schlesinger, J. J. (1987). Immunization with glycoprotein subunits of respiratory syncytial virus to protect cotton rats against viral infection. J Infect Dis 155, 1198–1204.[Medline]
Williams, J. V., Wang, C. K., Yang, C. F., Tollefson, S. J., House, F. S., Heck, J. M., Chu, M., Brown, J. B., Lintao, L. D. & other authors (2006). The role of human metapneumovirus in upper respiratory tract infections in children: a 20-year experience. J Infect Dis 193, 387–395.[CrossRef][Medline]
Received 15 April 2007;
accepted 31 May 2007.
This article has been cited by other articles:
|
|
 |
|
  S. Herfst, M. de Graaf, E. J. A. Schrauwen, L. Sprong, K. Hussain, B. G. van den Hoogen, A. D. M. E. Osterhaus, and R. A. M. Fouchier Generation of temperature-sensitive human metapneumovirus strains that provide protective immunity in hamsters J. Gen. Virol., July 1, 2008; 89(7): 1553 - 1562. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
