Immunization of macaques with formalin-inactivated human metapneumovirus induces hypersensitivity to hMPV infection

Available online at www.sciencedirect.com

Vaccine 25 (2007) 8518–8528

Immunization of macaques with formalin-inactivated human metapneumovirus induces hypersensitivity to hMPV infection Rik L. de Swart ∗ , Bernadette G. van den Hoogen, Thijs Kuiken, Sander Herfst, Geert van Amerongen, Selma Y¨uksel, Leo Sprong, Albert D.M.E. Osterhaus Department of Virology, Erasmus MC, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands Received 24 August 2007; received in revised form 27 September 2007; accepted 3 October 2007 Available online 26 October 2007

Abstract Human metapneumovirus (hMPV), a member of the family Paramyxoviridae, is an important cause of acute respiratory tract disease. In the 1960s, vaccination with formalin-inactivated paramyxovirus preparations – respiratory syncytial virus (RSV) and measles virus (MV) – resulted in predisposition for enhanced disease upon natural infection. We have produced a formalin-inactivated hMPV preparation (FI-hMPV), which was used to immunize young cynomolgus macaques. Six days after challenge FI-hMPV-primed monkeys had developed eosinophilic bronchitis and bronchiolitis, indicative of a hypersensitivity response. This study indicates that formalin-inactivated hMPV vaccines have the same propensity to predispose for immune-mediated disease as inactivated RSV and MV vaccines. © 2007 Elsevier Ltd. All rights reserved. Keywords: hMPV; Vaccination; Formaldehyde; Immunopathology; Paramyxovirus

1. Introduction Human metapneumovirus (hMPV) was first described in 2001 as the causative agent of acute respiratory tract infections (RTI) in hospitalized young children [1]. On basis of sequence information and genomic organization it was classified as a member of the family Paramyxoviridae, subfamily Pneumovirinae, genus Metapneumovirus [2]. The virus is globally distributed, and has been detected in 5–25% of patients hospitalized for RTI [3–6]. By the age of 5 years virtually everybody has experienced at least one hMPV infection [7]. Similar to most other respiratory viruses, hMPV-related disease shows a seasonal pattern with outbreaks in late winter or early spring in moderate climate zones [8]. The clinical symptoms are almost identical to those caused by respiratory syncytial virus (RSV), a member of the same virus family [3,6]. Like RSV, the most important risk groups for hMPVassociated severe disease are infants, immunocompromised patients and the elderly [7]. ∗

Corresponding author. Tel.: +31 10 4088280; fax: +31 10 4089485. E-mail address: [email protected] (R.L. de Swart).

0264-410X/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2007.10.022

The burden of disease caused by hMPV infections justifies development of intervention strategies. Vaccines designed to immunize infants will have to induce a primary immune response in the presence of maternal antibodies, whereas vaccines designed to immunize the elderly will need to boost waning immunity. Since hMPV and RSV are closely related, hMPV vaccine development can profit from previous experience with RSV vaccine candidates. In the 1960s a formalin-inactivated and alum-adjuvanted RSV vaccine (FI-RSV) was found to predispose infants for enhanced disease following natural RSV infection [9]. Patients developed high fever and severe pneumonia associated with cellular infiltrates, resulting in high hospitalization rates and even some fatalities. Studies in animal models demonstrated that FI-RSV induced an incomplete and unbalanced type of immunity dominated by T-helper type 2 (Th2) cells, while specific cytotoxic T-cells (CTL) were absent. Formalin inactivation links carbonyl groups to proteins promoting the induction of Th2 responses [10], which is further exacerbated by the use of alum as an adjuvant which has been associated with the induction of Th2 responses [11]. Pulmonary recall of these cellular immune responses during natural infection

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resulted in hypersensitivity responses [12,13]. In addition, vaccine-induced specific antibody responses proved to be of low avidity resulting in immune complex formation following natural infection, further contributing to the pulmonary hypersensitivity [14]. Vaccine-mediated enhanced disease was also observed for measles virus (MV), yet another member of the family Paramyxoviridae. In the 1960s a formalin-inactivated and alum-adjuvanted whole virus preparation (FI-MV) similar to FI-RSV was used to immunize infants. Vaccination initially conferred protection, but MV-specific antibody levels proved to be short-lasting. Some of the vaccinated children developed an enhanced disease syndrome referred to as atypical measles upon natural MV infection, which was associated with high fever, abnormal rash and pneumonia with cellular infiltrates [15]. The pathogenesis of this disease proved to be quite similar to that observed in FI-RSV vaccinated infants upon natural RSV infection [16–18]. The observations of vaccine-mediated enhanced disease for these two different paramyxoviruses raised the question whether the use of inactivated hMPV preparations to immunize infants would also predispose for hypersensitivity responses. Recently, Yim et al. described that immunization with formalin-inactivated hMPV predisposed cotton rats for enhanced pulmonary disease upon hMPV challenge [19]. We have immunized infant macaques with a formalin-inactivated whole virus preparation adjuvanted with alum (FI-hMPV), and evaluated virological, immunological and pathological responses upon hMPV challenge.

2. Materials and methods 2.1. Vaccine preparations A molecular clone of hMPV (strain NL/1/00, prototype virus for serotype A) [20] was grown in Vero-118 cells as previously described [21]. This virus strain consistently causes cytopathic effects (cpe) in this cell line, mainly characterized by induction of syncytia and rounding of cells between 4 and 7 days after infection. When the first cpe was observed, the cells were washed, and medium was replaced by Iscove’s Modified Dulbecco’s Medium (IMDM) without bovine serum albumin (BSA), trypsin, penicillin or streptomycin. At 75% cpe the medium was refreshed, and the following day supernatant was harvested and clarified by centrifugation (10 min, 1000 × g). The infected cells were freeze-thawed in 5 ml phosphate-buffered saline (PBS) at −80 ◦ C, clarified, and added to the supernatant. The resulting virus preparation was concentrated and purified by sequential ultracentrifugation: run 1 on a 60% sucrose cushion (5 ml, SW28 rotor, 27,000 rpm, 2 h, 4 ◦ C), run 2 on a 60% sucrose cushion (0.5 ml, SW41 rotor, 39,000 rpm, 2 h, 4 ◦ C) and run 3 on 30%/60% sucrose (10 and 0.5 ml, respectively, SW41 rotor, 39,000 rpm, 16 h, 4 ◦ C). The resulting interphase was harvested and dialyzed against TEN-buffer: 20 mM Tris–HCl,

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1 mM EDTA, 0.15 M NaCl, pH 7.8. The total protein content was determined using the BCA kit (Pierce), and the antigen was diluted to a concentration of 200 ␮g/ml. Formalin (37% formaldehyde, Merck) was added to a final formaldehyde dilution of 1:4000, and the antigen was incubated for 3 days at 37 ◦ C on a rotating device. Subsequently, aliquots were frozen at −80 ◦ C. Measles virus strain Edmonston was also grown in Vero118 cells in the same medium as used for hMPV, but without trypsin and BSA and with 1% fetal bovine serum (FBS) added. The virus was harvested, concentrated and purified as described for hMPV, dialyzed against TEN-buffer, diluted to a total protein concentration of 200 ␮g/ml, formalininactivated and frozen in aliquots at −80 ◦ C. A vaccine dose was prepared by mixing formalininactivated hMPV or MV antigen (50 ␮g per dose) with Alhydrogel 2.0%, kindly provided by Dr. E.B. Lindblad, Brenntag Biosector, Frederikssund, Denmark (0.85 mg aluminum per dose) and adding TEN-buffer to a final volume of 0.5 ml. The vaccine was incubated at room temperature for 1 h to allow adsorption of the antigen to the alum, and administered intra-muscularly. 2.2. Challenge virus The molecular clone of hMPV strain NL/1/00 was grown in Vero-118 cells as previously described [21]. At 80–90% cpe, cells were freeze-thawed at −80 ◦ C. Cells and supernatant were harvested and stored at −80 ◦ C with 25% sucrose. The resulting virus stock had a titer of 108 cell culture infectious dose-50 (CCID50 ) as measured by endpoint titration in Vero-118 cells (using spinoculation [22] and calculated according to the method of Reed and Muench [23]). In prior infection studies in macaques with this virus stock a dose of 104 CCID50 was found insufficient, while a dose of 106 –107 CCID50 resulted in infection as demonstrated by re-isolation and seroconversion [24]. In the present study a challenge dose of 107 CCID50 was used. A mock challenge stock was prepared by growing Vero118 cells in the same medium until a 100% monolayer was formed, freeze-thawing the flasks at −80 ◦ C, adding sucrose to a final 25% and freezing aliquots at −80 ◦ C. 2.3. Study design Twenty young cynomolgus macaques (Macaca fascicularis, mean age at first vaccination 8 months, range 5–16 months) were immunized three times (analogous to vaccination studies performed in the 1960s with FI-RSV and FI-MV) with FI-hMPV (groups 1 and 3), FI-MV (group 2) or injected with saline as a control (group 4), with time intervals of 4 weeks (see Table 1). Three months after the third vaccination the animals were challenged with hMPV (groups 1, 2 and 4) or a mock preparation (group 3). The challenge virus (107 CCID50 ) or the mock preparation were diluted to a final volume of 6 ml in PBS and

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Table 1 Study design Group number

Animal number

Sexa

Ageb

Vaccine

Challenge

1

1 2 3 4 5

M F M F F

6 7 8 10 13

FI-hMPV

hMPV

2

6 7 8 9 10

M F M F F

6 8 9 10 11

FI-MV

hMPV

3

11 12 13 14 15

M F M F F

7 8 10 5 16

FI-hMPV

Mock

4

16 17 18 19 20

F F F M M

6 6 8 9 13

Saline

hMPV

a b

M, Male; F, female. Age at first vaccination, in months.

administered intra-nasally (1 ml divided over both nostrils) and intra-tracheally (5 ml, inoculated just below the larynx). Six days after challenge all animals were euthanized by exsanguination and necropsies were performed. The study was approved by the Animal Ethics Committee, and was carried out in accordance with animal experimentation guidelines. 2.4. Virus-specific IgG ELISA IgG antibody responses to the hMPV and MV nucleoproteins were determined by indirect ELISA. High binding ELISA plates (Costar) were coated with an in-house produced baculovirus-produced recombinant nucleoprotein of hMPV or measles virus (kind gift of Dr. T.F. Wild, Lyon, France) at a concentration of approximately 50 ng/well in PBS (overnight, 4 ◦ C). Plates were washed and incubated with macaque serum samples diluted 1:100 in ELISA buffer (Meddens Diagnostics, Vorden, The Netherlands) supplemented with 5% normal goat serum (45 min, 37 ◦ C). Plates were washed again, and incubated with a peroxidase-labeled goat-anti-human IgG conjugate (DAKO, Glostrup, Denmark) (45 min, 37 ◦ C). Finally, the plates were developed using tetrametyl benzidine (TMB) as a substrate. Extinctions were read in an ELISA reader at 450 nm using a reference filter of 620 nm. 2.5. Virus neutralization (VN) hMPV-specific VN antibody titers were determined by endpoint titration as previously described [25], with some

modifications. Sera were heat-inactivated before testing (30 min, 56 ◦ C). Briefly, twofold serial serum dilutions starting at 1:8 were incubated with approximately 30 CCID50 hMPV. After 1 h at 37 ◦ C, the mixture was applied to Vero118 cell monolayers by spinoculation. After another hour at 37 ◦ C, the samples were removed, the cells were washed and subsequently incubated with medium without serum and with trypsin/BSA. Seven days later infected wells were identified by immunofluorescence. MV-specific VN antibody responses were determined as previously described [26]. For both viruses VN titers were defined as the reciprocal of the highest serum dilution resulting in complete neutralization (means of duplicate measurements). Each experiment included virus titrations of the working solution of the virus, using twofold dilutions, 10–100 CCID50 per well was considered acceptable. The upper and lower detection limits of the MV VN assay were 45 and 0.1 international units (IU) per ml, respectively. All samples were tested in the same assay. 2.6. Virus detection after challenge Virus isolation was performed in Vero-118 cells using spinoculation, in the absence of fetal calf serum and in the presence of 0.02% trypsin and 3% bovine albumin Fraction V (Invitrogen, Groningen, The Netherlands). The Vero118 cells had been grown in Iscove’s Modified Dulbecco’s Medium (Biowhittaker) supplemented with 10% fetal calf serum (Biowhittaker) and 2 mmol/l glutamine (Biowhittaker). RT-PCR was performed as described previously [27]. Briefly, RNA was isolated from 200 ␮l swab-material by use of the High Pure RNA Isolating kit (Roche Diagnostics) and eluted in 50 ␮l distilled water. hMPV genome copies were detected by Taqman real-time PCR (performed in triplicate) on 5 ␮l of isolated RNA from each sample, using a standard curve generated by RNA runoff transcripts of a PCR product [27]. The virus titer was expressed as genome copies per mg tissue or per ml transport medium from pharyngeal swabs. 2.7. Lymphoproliferation assay Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood using density gradient centrifugation, and frozen in aliquots at −135 ◦ C in medium supplemented with 20% heat-inactivated (HI; 30 min, 56 ◦ C) fetal bovine serum (FBS, Greiner Bio-One, Frickenhausen, Germany) and 10% dimethyl-sulfoxide. For proliferative assays cells were thawed and cultured overnight in RPMI-1640 medium (BioWhittaker, Verviers, Belgium) containing penicillin (100 U/ml; BioWhittaker), streptomycin (100 ␮g/ml; BioWhittaker), l-glutamine (2 mM; BioWhittaker) and 2-mercapto-ethanol (10−5 M; Merck KGaA, Darmstadt, Germany) supplemented with 10% FBS and 1% HI pooled macaque serum. The next day the cells were

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counted, plated in 96-well round-bottom plates (1.5 × 105 per well, 150 ␮l/well), and stimulated in triplicate with beta-propiolactone-inactivated Vero-118 cell antigen, hMPV antigen, or MV antigen at previously determined optimal concentrations. All three antigens were produced in identical medium on the same cells, and processed in a similar way. After 4 days of culture supernatants were harvested (100 ␮l per well), and fresh medium containing BrdU (10 ␮M) was added. The next day the cells were harvested, pooled per stimulation, and processed for FACSstaining according to the manufacturer of the BrdU kit (Becton-Dickinson, Erembodegem, Belgium). Results are shown as the percentage BrdU-positive cells in the CD3positive lymphocyte population (after gating on basis of FSC/SSC profile). The results shown are the means of two independent experiments for each day of sample collection. 2.8. Cytokine ELISAs The supernatants collected in the lymphoproliferative experiments described above were used for detection of IFN␥ and interleukin-13 (IL-13) using macaque-specific ELISA

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systems (U-CyTech, Utrecht, The Netherlands) using the manufacturer’s instructions. Results shown are the means of cytokine measurements in supernatants from two independent stimulation assays, measured separately. 2.9. Necropsies and pathology Gross necropsies were performed according to a standard protocol. Samples of nasal concha, trachea, primary bronchus, left lung (after inflation with 10% neutral-buffered formalin), tracheo-bronchial lymph node, axillary lymph node, spleen, heart, liver, kidney, stomach and ileum were fixed in 10% neutral-buffered formalin and embedded in paraffin. Sections were cut at 5 ␮m, stained with hematoxylin and eosin, and examined by light microscopy. For quantitative assessment of eosinophils in the bronchial epithelium, four hematoxylin-and-eosin-stained sections of lung (two of the cranial lobe, two of the caudal lobe) per macaque were evaluated without knowledge of their treatment. In each section, five high power fields (40× objective) containing bronchial epithelium were arbitrarily chosen and the numbers of eosinophils in the bronchial epithelium, from the apical side of the epithelial cells down to the basement membrane, were counted.

Fig. 1. Vaccination-induced virus-specific serum antibody responses. Antibodies specific for hMPV (upper panels) or MV (lower panels) were measured by indirect ELISA using plates coated with recombinant nucleoprotein (left panels) or by virus neutralization (right panels). Results are shown as the means ± standard error for groups 1 (black circles), 2 (grey squares), 3 (black triangles) and 4 (open diamonds). Time points of vaccination (V1, V2, V3) and challenge are indicated by arrows at the top of the upper plots.

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Fig. 2. Vaccination-induced lymphoproliferative responses. PBMC collected 2 weeks after the third vaccination (panels A–D) or at the moment of challenge (panels E–H) were stimulated in vitro with BPL-inactivated Vero-118 cell antigen, hMPV antigen, MV antigen or with medium as a control as indicated on the x-axis. After 4 days of culture supernatants were harvested, and fresh medium supplemented with BrdU was added. The next day the cells were harvested, after which T-cells that had incorporated BrdU were detected by FACS analysis. The bars indicate the mean percentage of positive cells per group, while responses of each individual animal are indicated by symbols: circle (animal 1), triangle down (animal 2), square (animal 3), diamond (animal 4) and triangle up (animal 5, same symbols used in all figures).

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Fig. 3. IFN-␥ (white bars) and IL-13 (grey bars) levels in supernatants of the cultures described in legend to Fig. 2. Bars indicated the mean cytokine levels per group, while symbols indicate the responses per animal (same symbols as in Fig. 2). See legend of Fig. 2 for experimental conditions.

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3. Results

3.3. Virus detection upon hMPV challenge

3.1. Serum antibody responses upon vaccination

Three months after the third vaccination the animals were challenged with hMPV (groups 1, 2 and 4) or with a mock preparation containing the same cells and medium as the challenge virus (group 3). As shown in Fig. 4, no protective effect of the FI-hMPV vaccination was demonstrated either by virus isolation (upper panel) or real-time RT-PCR (middle panel) in throat swabs collected on different time points after hMPV challenge. Six days after challenge all animals were eutha-

Twenty young macaques received three injections with FI-hMPV, FI-MV or saline (as indicated in Table 1), with intervals of 4 weeks. All animals vaccinated with FI-hMPV (groups 1 and 3) showed hMPV-specific (but not MVspecific) serum IgG and VN antibody responses (Fig. 1). In contrast, all animals vaccinated with FI-MV (group 2) showed MV-specific (but not hMPV-specific) serum IgG and VN antibody responses. No hMPV- or MV-specific antibodies could be detected in animals injected with saline. Antibody levels declined during the 3-month period between the third vaccination and the challenge, but at the moment of challenge (week 23) all FI-hMPV vaccinated animals still had detectable hMPV-neutralizing antibody titers.

3.2. Cellular immune responses upon vaccination Two weeks after the third vaccination hMPV-specific lymphoproliferative responses were detected in PBMC collected from animals vaccinated with FI-hMPV (groups 1 and 3), but not in animals vaccinated with FI-MV (group 2) or injected with saline (group 4) (Fig. 2, left panels). Similar responses were measured in PBMC collected on the day of challenge: in contrast to the specific antibody levels, the lymphoproliferative responses did not wane over time (Fig. 2, right panels). In some animals vaccinated with FIMV an apparently MV-specific lymphoproliferative response was detected, but background proliferation levels were much higher than those in the animals primed with FI-hMPV. A likely explanation for this difference is that the FI-MV vaccine contained a low level of FBS, which was also present in the culture medium used in these assays. This in contrast to the FI-hMPV vaccine, which was produced in serumfree medium. PBMC collected from macaques injected with saline only showed very low lymphoproliferation levels, which did not change after stimulation with hMPV or MV antigen (Fig. 2). The supernatants collected from the cultures described above contained low or undetectable levels of IFN-␥, but high levels of IL-13 (Fig. 3). The IL-13 levels largely paralleled the lymphoproliferative responses: hMPV-specific responses were detected in all animals of groups 1 and 3, while MVspecific responses were detected in some animals of group 2, the latter being confounded by high backgrounds in the control stimulations. No IL-13 was detected in the supernatant of any of the animals injected with saline. In contrast to the lymphoproliferative responses, the cytokine responses showed a pattern of declining levels between PBMC collected early after the third vaccination and PBMC collected on the days of challenge (Fig. 3, compare panels A–C with E–G).

Fig. 4. hMPV virus loads in throat swabs (panels A and B) or lung tissue (panel C) as detected by virus isolation (panel A) or RT-PCR (panels B and C). Bars represent the geometric meant responses of groups 1 (white), 2 (grey) and 4 (black). Animals of group 3 were mock challenged, and no hMPV-specific virus was detected (not shown). Symbols for responses of individual animals are identical as those in Figs. 1 and 2. Significant differences between the groups as measured by Student’s t-test are indicated by (* p < 0.05) or (** p < 0.01).

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Fig. 5. H&E staining of lung tissues collected 6 days after challenge. Panels A–C: bronchiole walls; panels D–E: alveoli. Representative examples are shown for animals in group 1 (panels A, D), group 2 (panels B, E) and groups 3 and 4 (panels C, F). Infiltration of eosinophils in the walls of the respiratory epithelium was seen in all five animals of group 1, in two out of five animals of group 2 and in none of the animals of groups 3 and 4. Eosinophilic alveolitis was only seen in one animal of group 1.

nized and necropsies were performed. In lung tissues of group 1 animals (immunized with FI-hMPV) a slight but statistically significant lower hMPV load was detected compared to group 4 animals (sham-immunized with saline). 3.4. Histopathology upon hMPV challenge All five animals of group 1 (primed with FI-hMPV and challenged with hMPV) had developed a superficial eosinophilic inflammation 6 days after challenge, affecting the walls of the nasal cavity (n = 4), trachea (n = 4), bronchi (n = 5), bronchioles (n = 5) and alveoli (n = 3). The histological changes were characterized by diffuse infiltration of eosinophils in epithelium and submucosa, disorganization of the epithelial architecture, and intercellular edema. One animal of group 1 (animal no. 3) had a multifocal interstitial pneumonia, characterized by flooding of the alveolar lumina by eosinophils, alveolar macrophages and fibrin, necrosis of alveolar epithelium, and type II pneumocyte hyperplasia (Fig. 5A and D). In the other animals no significant numbers of infiltrating cells other than eosinophils were observed. In group 2 (primed with FI-MV and challenged with hMPV), two of the five animals had similar eosinophilic inflammation in the airways from nasal cavity down to the bronchioles, but not in the alveoli (Fig. 5B and E). There was no histological evidence of eosinophilic inflammation in any animals of group 3 (primed with FI-hMPV and challenged with a mock preparation containing Vero-118 cell proteins) or group 4 (primed with saline and challenged with hMPV, see Fig. 5C and F). Quantification of eosinophil infiltration showed that

Fig. 6. Eosinophil numbers in bronchus walls 6 days after challenge. V, vaccine used for priming; C, challenge (either hMPV infection or mock challenge).

high numbers of eosinophils were present in all five animals in group 1, while none were detected in groups 3 and 4 (Fig. 6). Group 2 showed an intermediate pattern, with high numbers in two animals and low numbers in the remaining three.

4. Discussion In the present paper we have shown that immunization of young macaques with a formalin-inactivated hMPV preparation formulated in alum induced virus-specific humoral and cellular immune responses associated with production of

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Th2-cytokines. Upon challenge with hMPV 3 months after the third vaccination, all FI-hMPV-primed animals developed an eosinophilic tracheo-bronchitis, indicative of a hypersensitivity response. These responses were not observed in animals primed with FI-hMPV and challenged with a mock cell preparation, or in animals primed with saline and challenged with hMPV. However, a hypersensitivity response was also observed in two out of five animals that had been primed with FI-MV and were challenged with hMPV. The phenotype of the humoral and cellular immune response induced by vaccination with FI-hMPV was reminiscent of the immune responses previously described for macaques vaccinated with FI-RSV [13]. hMPV neutralizing antibody responses were detected in all FI-hMPV-vaccinated animals, albeit at low levels. In a previous study we showed that experimental infection of macaques with the same virus as used in the present study induced hMPV-specific VN antibody responses ranging between 100 and 10,000 [24], whereas in the present study vaccination-induced VN titers (measured using the same assay) stayed below 100. In the previous study we demonstrated that animals in which VN antibody titers had waned below a level of 100 were susceptible to challenge infection with the same dose as used here [24], which is in good accordance with the fact that we observed only limited protection in the FI-hMPV vaccinated animals in the present study. hMPV-specific T-cell responses were readily detected in all animals of groups 1 and 3. The implementation of a new non-radioactive technique in the present study did not allow direct quantitative comparison with our previous study [13]. However, comparison of cytokine levels in supernatants of PBMC cultures showed low or absent IFN-␥ and high IL-13 levels upon ex vivo hMPV antigen-stimulation, indicating the presence of specific cells with a Th2 phenotype. IL-13 has previously been identified as a central cytokine in the pathogenesis of asthma and allergic airway disease [28,29], and was also produced by vaccineinduced specific T-cells in the FI-RSV study in macaques [13]. The ex vivo responses detected in animals of group 2, which had been vaccinated with FI-MV, showed a similar phenotype but were not restricted to ex vivo MV antigen stimulated cultures. In contrast to the animals of groups 1 and 3, lymphoproliferative responses and IL-13 production were also detected upon stimulation with heterologous antigen, Vero-118 antigen or medium alone. The two vaccine preparations were prepared using identical protocols. The only difference was that MV was grown in medium supplemented with 1% FBS, while hMPV was grown in serum-free medium. In our hands it proved impossible to produce a MV stock in serum-free medium. Although the virus was purified by ultracentrifugation on sucrose gradients, the resulting antigen may still have contained FBS proteins, which upon injection with alum may have resulted in induction of FBSspecific T-cell responses. FBS has previously been described to be highly immunogenic, and a potential confounding factor in immunopathology studies [30,31].

Upon challenge with hMPV, all five FI-hMPV primed animals in group 1 developed an eosinophilic tracheo-bronchitis. One of these animals also developed a multifocal eosinophilic alveolitis. Eosinophilic tracheo-bronchitis was also observed in two out of the five animals primed with FI-MV and challenged with hMPV. No infiltration of eosinophils was seen in the animals of group 3, which had been primed with FI-hMPV and challenged with a mock preparation containing cellular proteins. This control group suggests that the responses seen in group 2 were not directed to cellular proteins. However, the responses are not directed to serum proteins, since the challenge virus was produced in serum-free medium. Due to the large phylogenetic difference between hMPV and MV [2] a role for cross-reactive T-cell epitopes is also considered unlikely: in the past we have actively searched for cross-reactive epitopes between members of different paramyxovirus genera, without success (R.L. de Swart, unpublished observations). We speculate that the responses in the two animals in group 2 were rather directed to cellular components, and that similar hypersensitivity responses were not detected in animals of group 3 because of the absence of a “danger signal” associated with active virus replication [32,33]. It is interesting to note that the two animals of group 2 that demonstrated infiltration of eosinophils in their airways also demonstrated relatively high lymphoproliferative and IL-13 responses in the ex vivo PBMC stimulation assays (see Figs. 2 and 3). We did not to use a purified virus stock for our challenge experiment, which might have prevented part of the observed non-specific response. Prior to the start of our study we reasoned that immune responses to non-viral antigens were not expected, because we used a purified virus preparation for vaccination. In previous immunopathology studies in animal experiments, experimental artifacts were described related to priming and recall of responses to non-viral antigens, for which these contaminants have to be present both in the vaccine and in the challenge preparation [30,31]. We therefore decided to use the same (non-purified) virus stock as used in our previous experiments [24]. The eosinophilic tracheo-bronchitis observed upon hMPV challenge in primed animals clearly indicates that FI-hMPV vaccination had induced hypersensitivity responses. The observation that eosinophils were mainly detected in nose, trachea and bronchi fits well with our previous observations in macaques that hMPV mainly replicates in ciliated epithelial cells [21]. Antigen production in the cells must have resulted in recall of the primed Th2-responses, which were unable to limit or clear the infection but instead resulted in mobilization of eosinophils to the site of virus replication. We did not include a control group that was naturally infected with hMPV, to assess if these animals would show develop eosinophilic tracheo-bronchitis upon hMPV challenge infection. However, unpublished observations from our RSV and measles models suggest that this would not be the case. In these experiments FI-RSV or FI-MV primed animals showed hypersensitivity responses upon challenge infection

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with RSV and measles virus, respectively. In both cases naive control animals did not, similar to the animals in group 4 of our current study. However, animals that had been experimentally infected with RSV three times with 4-week intervals (in parallel with the FI-RSV vaccinations), or animals that had been vaccinated with a live-attenuated measles vaccine, did not show infiltration of eosinophils in their airways, or any other signs of enhanced pulmonary disease (data not shown). The example of the RSV study suggests that the use of a nonpurified challenge virus does not explain the observations in the animals of group 2, since repeated infection with a nonpurified virus did not predispose for enhanced pulmonary disease in the RSV model, using the same challenge virus stock for priming and challenge infection. The observed infiltration of eosinophils would not necessarily result in clinically significant disease. In fact, the contribution of eosinophils in FI-RSV-mediated enhanced disease has been debated [34], and other immune-mediated phenomena such as pulmonary hyperresponsiveness may have had a more significant contribution to FI-RSV vaccinemediated enhanced disease observed in the 1960s [14]. However, in order to avoid the risk of vaccine-mediated enhanced disease for hMPV, vaccines targeted for use in infants should probably be based on alternative strategies, like the use of live-attenuated vaccines [35] or vector-based vaccines [36]. In contrast, for the elderly non-replicating whole virus or subunit vaccine candidates may probably be considered, as in this target group immunity to hMPV has already been primed, and vaccines may be expected to boost preexisting immunity. However, these vaccines would need to be more immunogenic than our FI-hMPV preparation, which might be achieved by using purified or recombinant glycoproteins formulated in new-generation adjuvants. The results we found in our macaque model were slightly different from recent observations in the cotton rat model as described by Yim et al. [19]. As opposed to our study, the FI-RSV preparation administered to cotton rats provided good lung protection. However, FI-hMPV-primed animals still developed enhanced pulmonary disease upon hMPV challenge, mainly characterized by accentuated peribronchiolitis, interstitial pneumonitis and alveolitis. The authors did not report infiltration of eosinophils, which may be explained by the fact that their FI-hMPV preparation was not adjuvanted with alum. However, in a previous report they emphasized the importance of the infiltration of neutrophils in enhanced pulmonary disease mediated by inactivated paramyxovirus vaccines, as these were also the major infiltrating cells in the lungs of the two children that died during the FI-RSV trial in the 1960s [34]. However, the most important common observation made in both studies is that vaccination with inactivated hMPV may predispose for enhanced pulmonary disease upon hMPV challenge. All animals appeared clinically normal in the present study. In a previous study, we reported that two our of seven FI-RSV-primed macaques developed severe hyperinflation 12 days after RSV challenge, and had to be euthanized [13].

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However, in the present study all animals were euthanized at earlier time points, making comparison of these responses impossible. In conclusion, the present study shows that immunization with formalin-inactivated hMPV not only fails to induce protective immunity, but also predisposes for hypersensitivity responses upon hMPV challenge infection.

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