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The Journal of Immunology, 2006, 176: 5848-5855.
Copyright © 2006 by The American Association of Immunologists

Human Metapneumovirus Elicits Weak IFN-{gamma} Memory Responses Compared with Respiratory Syncytial Virus1

Renée N. Douville*, Nathalie Bastien§, Yan Li{dagger},§, Pierre Pochard*, F. Estelle R. Simons*,{ddagger} and Kent T. HayGlass2,*,{dagger},{ddagger}

* Department of Immunology, {dagger} Department of Medical Microbiology, and {ddagger} Department of Pediatrics/Child Health, University of Manitoba, and § Canadian Science Centre for Human and Animal Health, Winnipeg, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Human metapneumovirus (MPV) is a recently discovered pathogen that causes repeated lower respiratory tract infections beginning in infancy. The prevalence, nature and control of human regulatory responses to MPV are unknown. In this study, we develop and optimize systems to evaluate MPV-driven cytokine responses. Using primary culture of human PBMC from previously exposed adults, MPV-stimulated responses were directly compared with those elicited by genetically and clinically similar respiratory syncytial virus (RSV). Intense IL-6 production was evident following culture with infectious or inactivated RSV. MPV elicited IL-6 responses averaging 3.5-fold more intense (p < 0.001). Virus-dependent expression of IL-11, IL-12, IFN-{alpha}, and other innate immunity cytokines differed little between MPV and RSV. When examining adaptive immunity, RSV infection elicited strong IFN-{gamma} responses by all 60 adults. In marked contrast, MPV elicited IFN-{gamma} in a lower frequency of adults (p < 0.002) and at levels averaging 6-fold weaker (p < 0.001). These Th1-dominated responses were CD4, CD8, CD86 dependent, and were closely paralleled by strong virus-driven IL-10 and CCL5 production. For MPV and RSV, Th2 (IL-5, IL-13) responses were sporadic, occurring in 10–40% of the population. Thus, MPV and RSV, although both ubiquitous and leading to very high levels of infection, seroconversion, and clinically similar presentation in the population, evoke distinct innate and adaptive T cell-dependent cytokine responses. Although both viruses yield Th1-dominated responses with strong IL-10 and CCL5 production, MPV restimulation results in markedly more robust IL-6 and significantly weaker adaptive cytokine responses, in both prevalence and intensity, than does RSV.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Virus infections are the primary cause of respiratory illness in humans. Of these, RNA viruses such as respiratory syncytial virus (RSV),3 human metapneumovirus (MPV), parainfluenza (PIV), and influenza virus are the most common pathogens associated with lower respiratory tract infections (1, 2). RSV is a particular health concern when infection is severe enough to lead to bronchiolitis, a condition that leads to hospitalization in 3% of infected infants and up to 16% in elderly patients with prior health complications (3, 4).

MPV is a recently discovered member of the Paramyxoviridae family (5) that is genetically (6) and clinically (7) similar to RSV. MPV is primarily a health concern in infancy, with symptoms that range from mild upper respiratory infections to severe lower respiratory infection requiring hospitalization. Most severe MPV infections are diagnosed as either bronchiolitis and/or pneumonitis (7, 8), with symptoms clinically indistinguishable from RSV infection (9). Like RSV, MPV is a cause of infantile bronchiolitis (10), although there are insufficient epidemiologic data to determine whether there is an etiologic association between acute MPV infection in early life and later development of asthma. However, mounting evidence points to MPV as a trigger of asthma exacerbations and pediatric wheezing syndromes (11, 12, 13, 14).

Extensive literature describes human humoral immune responses to RSV (15, 16, 17, 18, 19) and MPV (5, 19). Seropositivity usually develops during early childhood. Primary RSV infection induces Ab titers that wane over time and are boosted after multiple infections (20, 21, 22). A similar pattern of humoral responses appears to occur with MPV infections (5, 23). However, despite humoral immunity, symptomatic reinfection by RSV (24, 25, 26, 27) and MPV (7, 8, 9) occurs throughout life. In marked contrast to our increasing understanding of humoral immune responses to this recently discovered virus, nothing has been published examining the frequency or nature of immune regulation in MPV-specific responses.

The human immune response to RSV is seen as Th1-biased in clinical (28), ex vivo human PBMC (29, 30, 31), and murine (32, 33) models of infection. However, compared with other common upper respiratory tract infections such as adenovirus, rhinovirus, or influenza virus, RSV infection induces rather weak IFN-{gamma} responses (28, 34, 35). Increased severity of RSV infection is associated with decreased IFN-{gamma} levels in the airway (36, 37).

Currently, animal models have been used to study MPV-driven cytokine and chemokine responses (38, 39, 40). In BALB/c mice, MPV induces weak innate and Th1 responses, with Th2 cytokine production and IL-10 evident late in the course of infection (41, 42). Very little is known regarding the profile of MPV-dependent cytokine responses in humans (43, 44). In this study, we develop and optimize systems for the first comprehensive immunological study centered on the immune responses of humans to MPV. Using PBMC of healthy adults in short-term primary culture, MPV-specific innate and recall immune responses are assessed following in vitro restimulation, comparing infectious and inactivated virus. Our results clearly demonstrate that despite extensive genetic and clinical similarities between RSV and MPV, the immunologic responses they elicit differ markedly in the prevalence and intensity of antiviral IFN-{gamma} production. The implication of such differences in T cell-dependent immunity is that the weak antiviral recall responses expressed against MPV may contribute to the ability of this virus to cause recurrent infections throughout life.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Recruitment of participants and isolation of PBMC

Study approval was obtained from the University of Manitoba Faculty Committee on Use of Human Subjects in Research. Written informed consent was obtained from 63 healthy adults, 18–45 years old, with no evidence of current upper or lower respiratory tract infection. Blood was collected and PBMC were isolated by density centrifugation, counted (>95% viability by trypan blue exclusion), and used immediately for short-term primary culture. Plasma was collected and stored at –20°C.

rMPV protein ELISA

Cell lysates of baculovirus-rMPV nucleocapsid (N) protein (RV473-99/CAN99-80) and control wild-type baculovirus-infected insect cells were assayed for protein content using MicroBCA Protein Assay kits (Pierce). Using these Ags, an indirect ELISA was constructed to assess plasma Ab levels, as described (23). Briefly, baculovirus-expressed proteins were diluted to 3 µg/ml in PBS and 100 µl was coated to polystyrene flat-bottom microtiter plates (Nunc). Plates were washed, blocked, and human plasma samples were added at an optimal 1/100 and 1/400 dilution. Positive and negative serum control specimens were included in each assay to ensure reproducibility. Washed plates were subsequently incubated with peroxidase-conjugated anti-human IgG (1/25,000; Pierce) and developed using the 1-Step Turbo TMB substrate system (Pierce). To determine seropositivity, each individual’s N protein-specific IgG response was deemed positive if it was at least 2-fold greater than the mean optical density reading of the negative controls. In the present study, the median response was 6-fold above the threshold of detection.

Generation of viruses for cell culture

MPV strain CAN98-75 was cultured on LLC-MK2 cells and RSV strain Long on Hep-2 cells at 37°C in Eagle’s MEM supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), 30 µg/ml L-glutamine (Invitrogen Life Technologies). FCS (1%), and tosylsulfonylphenylalanylchloromethyl ketone trypsin (0.02%) was also added to the RSV and MPV growth media, respectively (Sigma-Aldrich). MPV virus isolates were cultured for a maximum of 21 days with weekly media change. Both MPV and RSV strains were titrated by the quantal assay 50% tissue culture-infective dose (TCID50) of Karber, performed in 96-well microtiter plates using 10-fold dilutions. Live virus was heat-inactivated for 30 min at 56°C to produce noninfectious virus for culture.

Primary cell culture

Freshly isolated PBMC were suspended in complete medium and 2.5 x 106 cells/ml were used for 200 µl cultures in 96-well U-bottom plates. Duplicate cultures were stimulated with MPV (104.3 TCID50/ml and heat-inactivated) or RSV (104.4 TCID50/ml and heat-inactivated). Streptokinase (Aventis Behring) was used at 5000 U/ml as a common recall Ag. In some experiments, anti-CD4 (2 µg/ml), -CD8 (4 µg/ml), -HLA-DR (2 µg/ml), -CD80 (5 µg/ml), -CD86 (1 µg/ml), isotype controls IgG1 (4 µg/ml) and IgG2a (10 µg/ml; BD Pharmingen), anti-HLA-ABC (10 µg/ml; Immunotech), and CTLA-4 Ig (5 µg/ml; a gift from Dr. P. Nickerson, University of Manitoba, Winnipeg, Manitoba, Canada), were used to evaluate activation requirements. Based on data obtained in preliminary experiments, culture supernatants were harvested following 1 or 6 days of culture, the time of peak MPV- and RSV-driven cytokine responses for the cytokines evaluated.

Human cytokine ELISAs

As a safety precaution, culture supernatants were UV irradiated for 1 h to inactivate residual infectious virus before ELISA analysis. Preliminary experiments (data not shown) demonstrated that this had no impact on the sensitivity or precision of the assays used to evaluate cytokine concentrations. Anti-cytokine capture and biotinylated detection Abs were purchased from BD Pharmingen, Endogen, Biolegend, or R&D Systems and recombinant cytokine standards from BD Pharmingen, Endogen, or PeproTech. They were used as previously described (45, 46). PBMC supernatants from duplicate cultures were assayed. All samples were evaluated in at least two assays, with the concentration in each supernatant calculated from a minimum of three points falling on the linear portion of titration curves that were calibrated against recombinant cytokine standards serially diluted on each plate. SEs typically ranged from 3 to 10%.

Flow cytometry

PBMC were cultured (2.5 x 106 cells/ml) in media, Staphylococcus enterotoxin B (SEB; Sigma-Aldrich), infectious MPV or RSV for 6, 12, 18, 24 h, 3 and 5 days. Washed cells (PBS with 1% BSA, 2 mM EDTA) were incubated 30 min on ice with fluorochrome-conjugated anti-CD3-PC5, CD4-FITC, CD8-FITC, CD25-PE, and CD69-PE and isotype-matched controls (BD Pharmingen or Biolegend). Cells were then washed, resuspended, and analyzed using FACSCalibur with acquisition and analysis performed on CellQuest Pro Software (BD Biosciences). The lymphocyte population within total PBMC was gated using forward and side scatter and CD3-PC5+CD4-FITC+ or CD3-PC5+CD8-FITC+ were selected and analyzed for CD69 and CD25 expression. SEB was used as a positive control for induction of activation markers, assessed both as percentage activated cells and intensity of marker expression. Also, CD69 and CD25 expression was analyzed as normalized mean fluorescent intensity (percentage of positive cells times mean fluorescent intensity), as described in Ref. 47 .

Statistical analysis

Associations between Ag-driven responses were determined using two-tailed Wilcoxon tests (paired, nonparametric data). Fisher’s exact test was used to compare differences in frequencies of responsive individuals. Normalized data in Fig. 6 were analyzed using a paired t test.


Figure 6
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  		FIGURE 6. Classical CD4+ T cell activation requires CD86 costimulation for human MPV and RSV-dependent IFN-{gamma} responses. MPV (A) and RSV (B) stimulated PBMC were cultured with neutralizing Ab to CD4, CD8, HLA-DR, or HLA-ABC. MPV (C) and RSV (D) infected PBMC were cultured with CTLA-4 Ig or neutralizing Ab to CD80 and CD86. Bars represent mean percentage (± SEM) of positive control, virus-stimulated IFN-{gamma} responses from five individuals. Values of p represent significant decreases in percentage of cytokine production compared with viral-driven response: ***, p < 0.001; **, p < 0.01). a, Anti-.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Seropositivity for MPV exposure is ubiquitous

Previous studies demonstrate that RSV exposure is ubiquitous and that the vast majority of adults are seropositive, with seroconversion occurring during the first two years of life (3, 24). To assess the prevalence of MPV seropositivity in this cohort of healthy adults, plasma were tested for Abs against the nucleocapsid protein of MPV (data not shown). MPV N protein-specific IgG demonstrated significant increases compared with background in each individual studied (n = 63, Wilcoxon p < 0.001), indicating that each individual had previously been exposed to MPV and developed an immune response. This finding is in agreement with recent studies indicating a high frequency of MPV seropositivity among healthy adults (5, 9, 23).

MPV elicits stronger innate immunity cytokine production than does RSV

To examine the frequency and nature of MPV-driven cytokine production in humans, we developed a short-term, in vitro primary culture system using PBMC isolated directly ex vivo from healthy adults. PBMC were cultured with infectious vs heat-inactivated MPV for 24 h, the time found in preliminary experiments with five subjects (data not shown) to yield maximum production of the innate immunity cytokines of interest. To compare these responses to phylogenetically related RSV, parallel cultures were set-up with live RSV at the same titer of infectious virus, and with heat-inactivated RSV. Incubation of PBMC with LLC-MK2 and Hep-2 cell lysates, as mock infection controls, did not induce detectable cytokine production for any of the readouts examined, demonstrating that cytokine responses elicited by these viruses were Ag dependent.

Strong IL-6 responses, with median values of ~800 pg/ml, were seen following stimulation with RSV. Similar levels were elicited by infectious and UV-inactivated virus. In contrast to RSV, MPV elicited IL-6 responses that averaged 200–300% more intense (Fig. 1). These increases were not dependent upon productive viral replication as inactivated virus yielded indistinguishable responses.


Figure 1
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  		FIGURE 1. Human MPV induces stronger IL-6 responses than RSV. Ag-dependent innate responses to RSV and MPV were compared with medium alone. IL-6 (A) and IL-11 (B) were measured by ELISA in supernatants from 24-h PBMC cultures. Black bars represent median responses from 14 to 41 healthy adults. Values of p represent significant differences in cytokine production: (***, p < 0.001; **, p < 0.01; *, p < 0.05).

 
In contrast to the robust IL-6 responses seen, only half of participants produced detectable IL-11 responses to either RSV or MPV (Fig. 1), and between a quarter and a third of individuals produced detectable IFN-{alpha} (data not shown). For these cytokines, no difference was evident between RSV- and MPV-driven stimulation. Other cytokines associated with innate immunity that play a role in promoting proinflammatory or Th1-like adaptive recall responses, such as IL-12, IL-1beta, TNF-{alpha}, or anti-inflammatory IL-10, were consistently undetectable at 24 h with either infectious or inactivated RSV or MPV (data not shown). Thus, based on production of innate immunity associated cytokines in a healthy adult population, MPV elicits similar, or for IL-6, markedly stronger responses than does RSV.

MPV-induced recall responses are distinct from those elicited by RSV

To evaluate adaptive cellular immune responses, fresh primary PBMC were cultured for 6 days with infectious vs inactivated MPV or RSV. Parallel cultures were established using streptokinase, a ubiquitous bacterial Ag that elicits T cell-dependent recall cytokine responses in most adults (48). As shown in Figs. 2 and 3, primary culture with streptokinase reveals readily detected type 1 (IFN-{gamma}) and type 2 (IL-13) recall responses, demonstrating the sensitivity of this experimental approach.


Figure 2
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  		FIGURE 2. Infectious human MPV and RSV induce Th1-biased recall responses. Ag-dependent recall responses to streptokinase (SK), live RSV, and live MPV, as compared with medium alone. IFN-{gamma} (A), IL-10 (B), CCL5 (C), and IL-13 (D) were measured by ELISA in supernatants from 6-day PBMC cultures. Black bars represent median responses from 60 individual healthy adults (•); ***, p < 0.001.

 

Figure 3
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  		FIGURE 3. Inactivated human MPV and RSV induce Th1-biased recall responses. Ag-dependent recall responses to streptokinase (SK), dead RSV, and dead MPV, as compared with medium alone. IFN-{gamma} (A), IL-10 (B), CCL5 (C), and IL-13 (D) were measured by ELISA in supernatants from 6-day PBMC cultures. Black bars represent median responses from 60 individual healthy adults (•); ***, p < 0.001.

 
RSV infection elicited strong IFN-{gamma} responses by all 60 adults in short-term culture. In marked contrast, stimulation with infectious MPV elicited detectable IFN-{gamma} responses in a significantly lower frequency of adults (74 vs 100%, Fisher’s p < 0.0002). Moreover, the median MPV-specific IFN-{gamma} response, while clearly substantial (9.6 U/ml, equivalent to 1100 pg/ml World Health Organization standard), was on average, 6-fold weaker than responses elicited by RSV. IL-13 and IL-5 (not shown) responses were weak in intensity and were detectable in ~10–40% of the population (Table I). Thus, no difference in the intensity or prevalence of type 2 immunity was evident when comparing MPV and RSV stimulation.


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  		Table I. Prevalence of infectious human MPV- and RSV-dependent responses in adultsa

 
Interestingly, Th1-biased memory responses to these viruses were paralleled by RSV-driven IL-10 and CCL5 responses (Figs. 2 and 3). MPV-driven IL-10 production was readily detected, but as seen with IFN-{gamma} responses, was on average 2.5-fold lower than RSV-elicited responses (p < 0.001). CCL5 was consistently produced in response to RSV, but was both less frequent (p < 0.0003, Table I) and markedly weaker (p < 0.001) in response to MPV stimulation.

Thus, this cohort demonstrated Th1-biased recall responses concomitant with virus-dependent IL-10 production to both MPV and RSV. For both viruses, the cytokine responses elicited were most pronounced upon stimulation with live virus. In contrast to its rapid capacity to elicit superior or equivalent innate immune responses, MPV consistently elicited IFN-{gamma}, IL-10, and CCL5 in recall responses more weakly than did RSV (Fig. 4). These increases in MPV stimulated IL-6 production, relative to RSV, and decreases in IFN-{gamma}, IL-10, and CCL5 were evident in almost all of the 48 individuals examined.


Figure 4
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  		FIGURE 4. Consistent differences in the intensity of human MPV-dependent responses compared with RSV. IL-6 (A), IFN-{gamma} (B), IL-10 (C) and CCL5 (D) were measured by ELISA. Paired RSV and MPV responses from 41 to 48 healthy adults (•) are shown. Values of p represent significant increases in cytokine production between RSV and MPV stimulation: ***, p < 0.001.

 
MPV-driven IFN-{gamma} production is mirrored by increased CCL5 and IL-10 production

Studies in IFN regulatory factor 1-deficient mice (49) and in fibroblast cell lines indicate that CCL5 expression is regulated by IFN-{gamma}. In this study, examination of human immune responses in primary culture to these two physiologically relevant viral stimuli demonstrates that CCL5 production is positively correlated with IFN-{gamma} (Fig. 5).


Figure 5
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  		FIGURE 5. IFN-{gamma}/CCL5 and IFN-{gamma}/IL-10 production during MPV and RSV recall responses are positively correlated. Infectious virus-dependent IFN-{gamma} and CCL5 responses in MPV (A) and RSV (B) and IFN-{gamma} and IL-10 responses in MPV (C) and RSV (D) stimulated cultures (day 6) were measured by ELISA. Black bars represent best-fit slope (r) of IFN-{gamma}/CCL5 or IFN-{gamma}/IL-10 pairs from 46 healthy adults (•). ***, p < 0.001; **, p < 0.01).

 
Given the range of individual responses seen throughout this population, and the fact that immune stimuli ranging from TLR ligands to endogenous and exogenous Ags usually demonstrate inverse production of IL-10 and IFN-{gamma}, we also examined the relationship between virus-driven IFN-{gamma} and IL-10 production to these respiratory viruses. Surprisingly, a strongly positive correlation between IFN-{gamma} and IL-10 levels was evident, with that for MPV-driven responses particularly strong (Fig. 5, r = 0.73, p < 0.001). Thus, individuals demonstrating recall responses to these respiratory viruses show concomitant increases in MPV- or RSV-dependent IFN-{gamma} coincident with proportional IL-10 and CCL5 induction.

T cell involvement in MPV- and RSV-specific cytokine production

To examine the role of CD4 and CD8 T cell populations in specific IFN-{gamma} responses, PBMC were cultured with live MPV or RSV in the presence of blocking anti-CD4, anti-CD8, anti-MHC class II or class I Abs (Fig. 6, A and B). As stated previously, MPV-specific responses (median 22 U/ml) were substantially less intense than those elicited by RSV (median 385 U/ml). However, each was dependent on both class I and II Ag presentation and activation of CD4 T cells. CD8 T cells make a substantive contribution to RSV-stimulated cytokine production but blocking the CD8 coreceptor had no detectable impact on MPV-driven IFN-{gamma} production. The potential role of IFN-{gamma} production by NK cells was not addressed; however, we note that the majority (>80%) of IFN-{gamma} induction in both RSV- and MPV-stimulated PBMC cultures was dependent upon CD4 T cell involvement and that IFN-{gamma} levels at day 1 were consistently undetectable (data not shown).

To assess the requirement for costimulation and putatively differential dependence on CD80 vs CD86 in this response, PBMC were virus stimulated in the presence and absence of neutralizing Abs to CD80, CD86, or with CTLA-4 Ig. Both RSV- and MPV-driven IFN-{gamma} production were clearly dependent on costimulation, with CD86 playing a pivotal role (Fig. 6, C and D).

In contrast, there was no evidence of T cell requirements for pneumovirus-specific IL-10 production (data not shown) as neither neutralizing Abs against CD4, CD8, MHC II, or MHC I, nor blocking of the CD80/86 costimulatory pathway, inhibited MPV- or RSV-driven IL-10 production.

MPV demonstrates reduced capacity to activate T cells relative to RSV

To extend the observation that MPV elicits weaker responses than does RSV in healthy adults, virus-stimulated PBMC were examined by flow cytometry to assess T cell expression of CD69 as an early marker and CD25 as a late marker of cellular activation. SEB-stimulated T cell populations were used as positive controls throughout. CD4+ and CD8+CD3+ T cells from RSV-stimulated cultures exhibited readily detected, and similar, increases in surface CD69 compared with unstimulated cells at 6–72 h (data not shown). Confirming the observations made for cytokine production, MPV induced weaker CD69 expression than did RSV through the time range examined, a finding evident for both CD4 and CD8 populations. Thus, at 12 h, the time of maximal CD69 expression, an average of 13% CD69+CD3+CD4+ T cells were detected in RSV-stimulated cultures vs 2% seen in MPV-stimulated cultures. In contrast to SEB, RSV and MPV stimulation failed to induce detectable increases in CD25 by either CD4+ or CD8+ T cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Despite increasing understanding of human serological responses to MPV (5, 19), virtually nothing is known of the presence, nature, or control of cytokine responses to this virus, or of the relationship between MPV- and RSV-driven responses in humans. In this study, we demonstrate that these viruses, both ubiquitous and leading to very high levels of infection and seroconversion in the population, elicit distinct innate and T cell-dependent cytokine responses. Primary culture of cells from a substantial cohort of healthy humans reveals that both viruses elicit classical CD4 T cell activation that is dependent on Ag-presentation and CD86-mediated costimulation. Among adults, MPV is a consistently stronger inducer of innate immunity associated IL-6, and a weaker inducer of IFN-{gamma}, IL-10, and CCL5 production. These differences in immune responses elicited by MPV vs RSV are evident whether measured by the frequency of responsive individuals or by the intensity of the cytokine response induced by virus exposure.

With the important exception of IL-6, MPV was a weaker inducer of cytokine production (IFN-{gamma}, IL-10, and CCL5) than was RSV. Interestingly, Guerrero-Plata et al. (50) in a recent detailed analysis of human myeloid and plasmacytoid dendritic cells derived from four to six healthy adults also found that these viruses elicit distinct patterns of cytokine responses. Interestingly, MPV-driven IL-6 responses by enriched DC were markedly weaker than those obtained upon RSV stimulation (50). This may be attributable to use of different viral isolates, comparison of isolated DC vs the global PBMC response or to the smaller numbers of subjects examined (n = 6 vs n = 63 here).

Use of dead RSV and MPV consistently demonstrates weaker cytokine responses (p < 0.05 to <0.001) than does exposure to live virus. Although we did not quantify the intensity of productive infection to these two viruses in culture across this population, the finding that stronger responses are observed upon active infection is consistent with prior observations (31, 50). The finding of Guerrero-Plata that RSV’s better ability to productively infect myeloid dendritic cells is linked with the increased strength of these antiviral cytokine responses underlines the role played by infection. At the same time, the overall pattern of responses seen for either virus (pro- vs anti-inflammatory; Th1 vs Th2) does not differ markedly following exposure to live or dead virus.

Two additional studies examine nonserological responses of humans to MPV infection. The first assessed levels of inflammatory cytokines in nasopharyngeal aspirates from 10 infants currently infected with MPV in comparison to cytokine levels in historic controls of RSV infection (43). They concluded that the pattern of local chemokine production seen with MPV differed from that seen for RSV. Laham et al. (44) comparing RSV-infected neonates with MPV-infected neonates demonstrated similar clinical manifestations during the course of active infection and, in general, higher levels of cytokine production in respiratory secretions of RSV-infected children. In this study, using 63 adults with histories but no clinical signs of ongoing MPV or RSV infection, primary culture of virus-stimulated PBMC demonstrates that MPV is a clearly superior stimulus for IL-6 production relative to RSV.

Why MPV’s capacity to elicit stronger IL-6 responses, and similar production of most other innate cytokines, is paralleled here by markedly weaker IFN-{gamma} (and IL-10, CCL5) production than RSV is unclear. Several mechanisms of IFN-{gamma} inhibition have been described in RSV infection, such as inhibitory viral proteins, regulatory effects on dendritic cells, and inhibition of immune memory (51, 52, 53, 54, 55). RSV nonstructural (NS1 and NS2) proteins can inhibit production of type 1 IFNs (IFN-{alpha}/IFN-beta), and also have key roles in down-regulating activation of APCs (51, 53, 56), thereby potentially diminishing T cell-dependent antiviral responses. One obvious disparity between the Pneumovirinae species is that MPV’s viral genome does not code for these NS proteins (5). This initially led us to anticipate that MPV-driven IFN-{gamma} responses would be stronger, not weaker, than those elicited by RSV. Given that MPV differs from RSV in lacking NS proteins, yet the IFN-{gamma} responses seen are weaker, the difference is clearly not attributable to this mechanism.

Alternately, the weak anti-MPV recall response, relative to those seen with many other respiratory viruses, may be attributable to the initial pattern of innate immunity that develops. MPV is far superior to RSV at inducing IL-6. IL-6 has been shown to inhibit Th1 differentiation and suppress IFN-{gamma} expression by direct effects on T cells (reviewed in Ref. 57). Naive T cells primed in the context of IL-6 are unable to differentiate into high IFN-{gamma}-producing effector cells (58). Thus, we speculate that during natural MPV infection in vivo, MPV-specific T cells may be primed in a milieu that leads to weaker primary and recall antiviral responses than are seen for RSV, a weaker inducer of IL-6 synthesis. Such studies are best approached in longitudinal analyses of deliberate infection of mice or human models (59).

Weaker Th1 responses may reflect lower frequencies of responding memory cells in individuals, as suggested by the substantial decrease in the frequency of individuals in the population with detectable IFN-{gamma} recall responses when comparing MPV to RSV. We tested whether this difference was related to activation of T cells by these viruses, however, no significant expression of activation markers on CD4 and CD8 T cells were detectable in MPV- or RSV-stimulated cultures. Minimal differences in frequency of CD69+ T cells were observed in RSV vs MPV stimulation, but CD25+ T cell frequency did not significantly differ. Others have found that circulating RSV-specific CD8 T cells are found at a lower frequency than influenza-specific cells (60), and their IFN-{gamma} recall responses are substantially lower (35). This may be partially explained by the fact that natural reinfection by RSV does not lead to long-term expansion of the RSV-specific T cell compartment in humans (61). Murine models of RSV infection also demonstrate impaired development and sustainability of memory CD8 T cells (55). It is presently unclear whether the lower MPV-dependent IFN-{gamma}, IL-10, and CCL5 responses seen reflect a lower prevalence of virus exposure in the community, hence fewer booster infections, or conversely, whether lower numbers of clinical reinfections (7, 9, 13) are attributable to IFN-{gamma}-, IL-10-, and CCL5-independent mechanisms of host resistance. In this study, the novel finding of positive correlation between IFN-{gamma}/IL-10 and IFN-{gamma}/CCL5 levels in both RSV- and MPV-stimulated PBMC suggests a mechanism governing the overall strength of antiviral response occurs in each individual.

The relevance of virus-stimulated cytokine production to clinical outcome is made explicit by strong associations between decreased levels of IFN-{gamma} and IL-10 production with increased severity of RSV infection (36, 37, 62). Similarly, it will be important to determine the extent to which the weaker capacity of MPV to induce IFN-{gamma} and IL-10 in vitro may relate to symptom severity and clinical outcomes during infection. Direct comparison of laboratory confirmed MPV, RSV, and influenza infections in families demonstrates that despite a lower prevalence of infection, MPV is associated with increased wheezing, asthma exacerbations, medical visits, and absenteeism (13). Thus, our data suggest a possible immunologic explanation for the greater symptoms and socioeconomic impact of MPV infection compared with RSV and influenza infections (13). Moreover, weak antiviral responses to MPV may be a contributing factor to the epidemiologic observations of increased asthma exacerbations. Consequently, we recently initiated studies to investigate putative differences in the nature and intensity of MPV vs RSV stimulated recall response in individuals with different clinical phenotypes.

The present study provides the first comprehensive overview of the cell-mediated immune response to MPV in humans. Despite extensive genetic and clinical similarities between these viruses, the immune response to MPV is characterized by stronger IL-6 and lower frequency, weaker intensity, IFN-{gamma}, IL-10, and CCL5 cytokine production in the population than is seen to RSV. These data demonstrate that the recall immune response to MPV is more limited that that of other typical respiratory viruses, and suggest a mechanism that may underlie the life-long, typically symptomatic reinfection with this virus (7, 8, 9).


   Acknowledgments
 
We thank S. Goritz, R.N., T. Thottingal, and B. Stefura for technical assistance and M. Cheang for assistance with statistical analysis.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Canadian Institutes for Health Research (CIHR). R.N.D. holds a Manitoba Health Research Council studentship, the Mindel and Tom Olenick Award in Immunology, and is a trainee member of the CIHR National Training Program in Allergy and Asthma. K.T.H. holds the Canada Research Chair in Immune Regulation. Back

2 Address correspondence and reprint requests to Dr. Kent T. HayGlass, Department of Immunology, University of Manitoba, 603 Basic Medical Sciences Building, 730 William Avenue, Winnipeg, Manitoba R3E 0W3, Canada. E-mail address: hayglass{at}ms.umanitoba.ca Back

3 Abbreviations used in this paper: RSV, respiratory syncytial virus; MPV, metapneumovirus; TCID50, 50% tissue culture-infective dose; SEB, Staphylococcus enterotoxin B; NS, nonstructural. Back

Received for publication November 17, 2005. Accepted for publication February 10, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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