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. Author manuscript; available in PMC: 2015 Jul 1.
Published in final edited form as: Virology. 2014 May 29;0:23–33. doi: 10.1016/j.virol.2014.05.004

The Neutralizing Capacity of Antibodies Elicited by Parainfluenza Virus Infection of African Green Monkeys is Dependent on Complement

Anne E Mayer 1, John B Johnson 1, Griffith D Parks 1,*
PMCID: PMC4101032  NIHMSID: NIHMS597599  PMID: 25010267

Abstract

The African Green Monkey (AGM) model was used to analyze the role of complement in neutralization of parainfluenza virus. Parainfluenza virus 5 (PIV5) and human parainfluenza virus type 2 were effectively neutralized in vitro by naïve AGM sera, but neutralizing capacity was lost by heat-inactivation. The mechanism of neutralization involved formation of massive aggregates, with no evidence of virion lysis. Following inoculation of the respiratory tract with a PIV5 vector expressing HIV gp160, AGM produced high levels of serum and tracheal antibodies against gp120 and the viral F and HN proteins. However, in the absence of complement these anti-PIV5 antibodies had very poor neutralizing capacity. Virions showed extensive deposition of IgG and C1q with post- but not pre-immune sera. These results highlight the importance of complement in the initial antibody response to parainfluenza viruses, with implications for understanding infant immune responses and design of vaccine strategies for these pediatric pathogens.

INTRODUCTION

The complement system is an important component of the innate immune response to viruses. Complement (C’) antiviral functions include a large number of activities, including recognition of viruses and virus-infected cells, direct neutralization of virus infectivity, recruitment and stimulation of leukocytes at sites of infection, phagocytosis by immune cells, and activation of antiviral T and B cells (Blue et al., 2004; Gasque, 2004; Kemper and Atkinson, 2007). Likewise, viruses employ mechanisms to limit C’ functions (e.g., Blue et al., 2004; Johnson et al. 2012). The balance between C’ effectiveness and virus inhibition of C’ can have important implications for viral pathogenesis and dissemination (Delgado and Polack, 2004; Morrison et al., 2007, Stoermer and Morrison, 2011). C’ can also directly impact adaptive immunity (Carroll, 2004; Kemper and Atkinson, 2007) and can influence the quality of anti-viral antibody responses (Pierson et al., 2008). The overall goal of the work described here was to determine the contribution of C’ to the neutralizing capacity of antibodies elicited by respiratory tract infection of nonhuman primates with parainfluenza virus.

The C’ proteolytic cascade can be initiated through three main pathways: the classical pathway, lectin pathway and alternative pathway (Gasque, 2004; Roozendaal and Carroll, 2006). Activation of the classical pathway typically involves binding of the C1q component to virus-antibody complexes. Human Immunodeficiency Virus (HIV; Ebenbichler et al., 1991) and vesicular stomatitis virus (VSV; Beebe and Cooper, 1981) are known to activate the classical pathway. The lectin pathway is activated through recognition of carbohydrate signatures on viral glycoproteins by the cellular mannan-binding lectin (MBL). This is an important pathway in the pathogenesis of Ross River Virus (Gunn et al., 2012) and in the opsonization of influenza virus (Hartshorn et al., 1993). Compared to activation of the classical and lectin pathways, the signals that activate the alternative pathway are less well understood, but they are thought to involve recognition of foreign surfaces by an antibody-independent mechanism (Gasque, 2004; Pangburn et al., 1981).

Parainfluenza virus 5 (PIV5), human parainfluenza virus 2 (HPIV2) and mumps virus (MuV) are closely-related negative strand RNA viruses belonging to the rubulavirus genus of the paramyxovirus family (Lamb and Parks, 2013; Parks et al. 2011). Prior work has shown that the rubulavirus attachment protein (Hemagglutinin-Neuraminidase; HN) and the fusion protein (F) can both contribute to activation of the alternative pathway (McSharry et al., 1981; Hirsch et al., 1986; Johnson et al., 2008; 2013). For PIV5 and MuV, the extent of alternative pathway activation is directly related to the loss of sialic acid on particles due to the presence of neuraminidase activity in the HN protein (McSharry et al., 1981; Hirsch et al., 1986). Furthermore, the rubulavirus F protein can dictate which arm of the C’ pathway is activated. This was evident by our recent finding that a single point mutation in the ectodomain of the PIV5 F protein led to increased fusion activity, but also led to enhanced binding of IgG contained in normal human sera (NHS) and a subsequent shift in C’ activation from the alternative to the classical pathway (Johnson et al., 2013).

Once activated, C’ components are capable of direct neutralization of viruses, through mechanisms that can include aggregation or virion lysis (Blue et al., 2004; Stoermer and Morrison, 2011). In addition, C’ can enhance the neutralizing capacity of antibodies (Mehlop et al., 2009). For HPIV2, our prior results demonstrated very high levels of neutralizing antibody in NHS (Johnson et al, 2008), making the contribution of C’ to neutralization difficult to analyze. In addition, repeated exposure to parainfluenza virus as infants (Karron and Collins, 2013) and the use of adult NHS in neutralization assays makes it difficult to determine the role of C’ in the antibody function following the very first exposure at an early age to human parainfluenza virus infection. By contrast, we have previously shown in reconstitution experiments that PIV5 is neutralized through pathways that are highly dependent on the alternative C’ pathway (Johnson et al., 2008). These mechanisms are either independent of antibody or involved antibodies in NHS that are only highly effective when coupled with C’.

Given the importance of understanding the initial immune response to parainfluenza virus infections, we have examined the role of C’ in a primary PIV5 respiratory tract infection of African green monkeys (AGM), an increasingly important model system for understanding primate immunology (Messaoudi et al., 2011). The animals used in this study were part of a multigenerational, pedigreed, and genotyped Vervet Research Colony (VRC) at the Wake Forest University Primate Center (described in Jasinska et al., 2012). A powerful aspect of using this AGM system lies in the ability to measure the effect of C’ on parainfluenza virus immunity in both naïve and vaccinated animals with immune systems that are closely related to that of humans. We show that respiratory tract infection of AGM with PIV5 elicits high levels of antibodies to viral antigens. However, these antibodies have poor inherent neutralizing capacity and are highly dependent on C’ pathways. Our results have implications for including C’ pathways as a major contributing factor in analysis of the potency of immune responses to parainfluenza virus infections and vaccine vectors.

RESULTS

Normal monkey serum has potent neutralizing capacity against PIV5 and HPIV2

To determine the ability of normal African Green monkey serum (NAGS) to neutralize PIV5 in vitro, serum samples were collected from 20 AGM and used in neutralization assays with 100 PFU of PIV5-GFP. After 1 h at 37°C, remaining infectivity was determined by plaque assay. As shown in Fig. 1A for representative sera from eight individual monkeys, a 1/20 dilution of NAGS reduced PIV5 infectivity to undetectable levels. In contrast, HI AGM serum was ineffective in neutralization. Titration experiments (Fig. 1B) showed effective neutralization of PIV5 by at least a 1:160 dilution of NAGS, but HI serum had no neutralizing capacity. Similar results were seen with neutralization of HPIV2 by NAGS (Fig. 1C). These results indicate that in the absence of C’, NAGS from the WFU VRC have low capacity to neutralize these two parainfluenza viruses.

Figure 1. NAGS has potent C’-mediated neutralizing capacity against parainfluenza virus.

Figure 1

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A) One hundred PFU of PIV5-GFP were incubated for 1 h at 37°C with media alone (control Ctr; left gray bar) or with a 1:20 dilution of either NAGS (striped bars) or heat inactivated serum (HI, black bars). Remaining infectivity was determined by plaque assays. Numbers on the x-axis indicate individual animal numbers. Results represent the average of four to six assays, with error bars representing standard deviations. (*) no plaques were detected in these samples. B and C) One hundred PFU of PIV5-GFP (panel B, animal 1465) or of HPIV2 (panel C, animal 1484) were incubated with the indicated fold dilutions of NAGS or HI monkey serum as described for panel A and remaining infectivity was determined by plaque assay. D) Purified PIV5-GFP was incubated alone (left panel; 49,000X) or with a 1:20 dilution of NAGS (right panel; 18,000X) for one h at 37°C and then samples were placed on grids for analysis by electron microscopy. As a control for virion lysis, VSV was treated with normal human serum for 15 min as described previously (Johnson et al., 2012) before analysis by EM. The size of bars is indicated.

C’-mediated neutralization with NAGS could occur by lysis of virion particles (e.g. as seen with VSV; Johnson et al., 2012) or by formation of large aggregates (e.g., PIV5; Johnson et al, 2008). To determine the mechanism of PIV5 neutralization by NAGS, purified virus was incubated in solution at 37°C alone or with a 1:20 dilution of NAGS before applying to a grid for analysis by electron microscopy. As shown in Fig. 1D, treatment of PIV5 particles with NAGS resulted in the formation of massive aggregates (note scale bar in panels), with little evidence of virus lysis. As a control, treatment of vesicular stomatitis virus with human sera resulted in virion lysis as evident by the detection of nucleocapsid structures (Fig. 1D). Thus, like human sera (Johnson et al. 2008), the potent C’-dependent neutralization of PIV5 by NAGS is through a mechanism involving aggregate formation.

Antibodies do not contribute to C’-mediated neutralization of PIV5 by naïve NAGS

To determine if the animals used in this study had detectable levels of anti-PIV5 antibodies, mock infected or PIV5-infected A549 cells were analyzed for cell surface staining by NAGS. As shown in Fig. 2A, the positive control polyclonal anti-PIV5 mouse serum gave strong cell surface staining on PIV5 infected cells. As shown for one representative animal (Fig. 2A; animal #1484), the NAGS from animals used in this study showed no significant staining on PIV5-infected cells compared to mock infected controls. Using western blotting of lysates from mock infected and PIV5-infected cells, sera from a few animals showed very low level reactivity against N protein (star, Fig. 2B). However, similar to the results from the immunofluorescence analysis, there was no detectable signal for reactivity against the viral glycoproteins.

Figure 2. Anti-PIV5 antibodies do not contribute to C’-mediated neutralization of PIV5 by NAGS.

Figure 2

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A) A549 cells were mock infected or infected at an moi of 20 with PIV5. At 20 h pi, cells were analyzed for surface staining by immunofluorescence using mouse polyclonal anti-PIV5 serum or NAGS (animal 1484) as described in Materials and Methods. B) Lysates from A549 cells that were mock infected (M lanes) or infected (I lanes) with PIV5-GFP were analyzed by western blotting using NAGS from three representative AGMs. Control blots that were probed with mono-specific rabbit antisera to NP, P or M proteins served as markers for the position of viral proteins. Star in I lane, animal 1796 denotes weak reactivity with viral N protein. C) One hundred PFU of PIV5-GFP were incubated for 1 h at 37°C with media alone (control Ctr; left gray bar) or with a 1:20 dilution of either NAGS alone or with NAGS that had been depleted of antibodies by treatment with Protein G-sepharose. NAGS treated with sepharose alone served as a control. Remaining infectivity was determined by plaque assay. Results represent the average of four assays, with error bars representing standard deviations.

To directly test the role of antibodies in PIV5 neutralization by naïve animals, NAGS was depleted of IgG by treatment with Protein G-sepharose or with sepharose alone as a control and then tested for their capacity to neutralize PIV5-GFP in vitro. As shown in Fig. 2C, a 1:20 dilution of NAGS was equally effective in neutralizing PIV5-GFP when left untreated or following treatment with Protein G-sepharose or control sepharose lacking Protein G. Together, these data support the proposal that the animals used in this study lack substantial levels of antibodies to the PIV5 glycoproteins which could contribute to neutralization, and that the C’-mediated neutralization shown above in Fig 1 is through an antibody-independent mechanism.

A recombinant PIV5 expressing HIV gp160 elicits strong serum antibody titers to PIV5 antigens and gp120

To determine the ability of a PIV5 vector to elicit antibody responses to viral and vector-encoded antigens in AGM, PIV5 was engineered to express the HIV IIIB gp160 protein as an additional gene between HN and L (Fig. 3A). Cells infected with the PIV5-gp160 vector expressed high levels of gp160 as detected by western blotting (Fig. 3B). Analysis of multi-step growth in tissue culture cells showed that the PIV5-gp160 virus grew slightly slower than the control PIV5-GFP (Fig. 3C), but this difference was less apparent in analysis of single step growth (high moi).

Figure 3. A recombinant PIV5 engineered to express the HIV gp160 protein.

Figure 3

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A) The PIV5 genome is shown schematically with addition of the HIV-1 gp160 gene between HN and L as described previously (He et al., 1997). le and tr; leader and trailer. B) MDBK cells were mock infected (M lane) or infected (I lane) at an moi of 25, and cell lysates prepared at 18 h pi were analyzed by western blotting for NP and P (bottom panel) or for gp160 expression (top panel). C) MDBK cells were infected with the indicated viruses at an moi of 25 or 0.05 and virus titer was determined by plaque assay at 24 h pi (high moi) or at the indicated days (low moi). Error bars represent standard deviations.

To determine the capacity of PIV5-gp160 to elicit antibody responses, four adult 3 yr old AGM were innoculated (in two separate experiments using two animals each) by both the intranasal and intratracheal routes with 108 PFU of purified virus. Tracheal washes were obtained on d4 and d8, and serum was collected on d7 and d14 post inoculation. Throughout the timecourse of the experiment, animals showed no signs of overt illness, as evidenced by the lack of significant rise in temperature, no loss of cognitive function and the absence of labored breathing (data not shown). Analysis of tracheal wash fluid by TCID50 assay did not reveal detectable virus shedding at any time tested.

Pre-immune and d14 post-innoculation sera were analyzed by ELISA for anti-PIV5 antibodies as described previously (Johnson et al., 2008). As shown by the representative data for monkeys 1515 and 1536 in Fig. 4A, inoculated AGM generated anti-PIV5 IgG and IgM levels that were detectable above that seen with control preimmune sera out to 1:200,000 and 1:12,800, respectively. Titers expressed as the reciprocal of the dilution of sera giving 50% of maximum absorbance ranged from 31,000 to 70,000 for IgG and 2,200 to 4,800 for IgM (Fig. 4B). Similarly, serum antibodies against gp120 were detected above that seen with a control animal serum, although titers were not as high as that seen against PIV5 antigens and were less consistent between animals (Fig. 4A).

Figure 4. ELISA titers of AGM serum anti-PIV5 antibodies.

Figure 4

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A) Pre- and post-infection sera collected from infected AGM were tested by ELISA for the presence of anti-PIV5 IgG (left panel) and IgM (middle panel). Alternatively, post-infection sera were tested for IgG specific for gp120 (right panel), with serum from an AGM that did not receive PIV5-gp160 (animal 1152) serving as a control. Representative data are presented from animals 1515 and 1536. B) Final titers of IgG, IgM were calculated from dilutions of post-immune sera. C) Tracheal washes were collected from animals 1515 and 1536 on d4 and d8 post innoculation with PIV5-gp160. Samples were analyzed by ELISA for the presence of IgG specific for PIV5 or gp120. Control tracheal wash was collected from an animal that had not been exposed to PIV5-gp160.

Tracheal lavage samples from the inoculated AGM were tested for the presence of mucosal-associated anti-PIV5 IgG and IgM. As shown for animals 1515 and 1536 in Fig. 4C, levels of IgG at d4 post inoculation were detectable above control levels, but were substantially higher at d8 post inoculation. Tracheal lavage samples also contained anti-gp160 IgG (Fig. 4C) and IgM (not shown), although the titers were apparently much lower than that of the anti-PIV5 antibodies. These data indicate that innoculation of the AGM respiratory tract with PIV5-gp160 elicits strong systemic and mucosal antibody responses.

Effective neutralization of PIV5 with post-infection AGM sera requires intact C’ pathways

The capacity of AGM antibodies to neutralize PIV5 was tested by incubating dilutions of pre- and post-infection sera with PIV5-GFP and determining remaining infectivity by plaque assay. In addition, HI sera was analyzed to define the role of C’ in neutralization. As shown in the example in Fig. 5A for sera from animal #1690, pre-immune and d7 sera were effective in neutralization ~80% of infectivity at a 1:40 dilution, but was no longer effective after HI treatment. By contrast, d14 serum was highly effective in neutralizing PIV5, with no detectable infectivity seen out to a 1:200 dilution. Unexpectedly, HI removed much of the potency of neutralization by post-immune sera, since a 1:200 dilution of HI serum only neutralized ~20% of infectivity. Similar results were seen for all four PIV5-innoculated animals. For example, as shown in Fig. 5B, d14 post-immune sera from monkeys #1515 and #1536 were highly effective in neutralizing PIV5 out to at least a 1:200 dilution. However, after HI treatment both of these post-immune sera were only capable of substantial neutralization out to ~1:20. When higher dilutions of d14 post-immune sera were tested (Fig. 5C), PIV5 was neutralized at dilutions greater than 1:800. Final titers expressed as the reciprocal of the dilution of sera giving 50% neutralization ranged from 684, 842, 728 and 819 for animals 1515, 1536, 1685 and 1690, respectively. Together, these data indicate that the potency of d14 anti-PIV5 neutralizing antibodies is greatly reduced when sera are HI, indicating a high dependence on active C’ pathways.

Figure 5. Effective neutralization of PIV5 with post-infection AGM sera requires intact C’ pathways.

Figure 5

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A and B) PIV5-GFP was incubated with PBS (control, Ctr, gray bar) or with the indicated dilutions of normal or HI preparations of pre-immune, d7 post-immune or d14 post-immune sera from animals 1690 (panel A) or 1515 and 1536 (panel B). Remaining infectivity was determined by plaque assay. Results represent the average of four assays, with error bars representing standard deviations. (*)no plaques were detected in these samples. C) PIV5-GFP was incubated with the indicated dilutions of d14 post-immune sera from the four AGM and samples were analyzed as described for panels A and B.

C1q is part of the C1 complex, which is necessary for and specific to the classical pathway. To test the hypothesis that post-immune AGM serum utilizes the classical pathway to neutralize PIV5, purified PIV5 particles were incubated with either pre-immune or post-immune serum. C1q and IgG deposition were each detected using gold bead labeling. PIV5 particles treated with pre-immune serum had no detectable IgG or C1q deposition (Fig. 6A). In contrast, high levels of both IgG (6 nm beads; small arrow) and C1q (12 nm beads; white arrow) were detected on the surface of PIV5 particles treated with post-immune sera (Fig. 6B). These data support the proposal that antibodies elicited at d14 post-infection are highly dependent on C’ for PIV5 neutralization, and proceed through the classical antibody-triggered pathway. This finding also confirms that pre-immune NAGS does not neutralize by the classical pathway, as there was no Ig or C1q deposition on virus particles treated with pre-immune serum.

Figure 6. C1q and IgG deposition on purified PIV5 after treatment with pre- and post-immune sera.

Figure 6

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A and B) Purified PIV5-GFP was treated with a 1/20 dilution of either pre- (panel A) or post-immune (panel B) monkey sera. Samples were subsequently treated with gold-conjugated anti-C1q (12 nm beads) and anti-human IgG (6 nm beads). Samples were analyzed by EM at a magnification of 68,000X. C and D) Purified PIV5-GFP was incubated with a 1:20 dilution of post-immune sera on ice for 1 min (Panel C; 49,000X) or for 15 min at 37°C (panel D; 18,000X) and then analyzed by electron microscopy. The size of bars is indicated. The black box in panel D indicates the area expanded in the 0.5 um image.

To determine the mechanism of neutralization by post-immune AGM serum, purified PIV5-GFP was treated with post-immune sera and at different times analyzed by EM. As shown in Fig. 6C for a representative monkey sample, serum-treated virions held on ice showed a mixture of dispersed particles as well as some aggregation. However, after as little as 15 min at 37°C, virions were only seen in massive aggregates with very little evidence of virion lysis (see size of bar in Fig. 6D, and expanded view of aggregate, Fig 6E). Thus, post-immune sera (Fig. 6) and pre-immune sera (Fig. 1) both neutralize PIV5 by aggregation, with the major difference being the more rapid aggregation seen by post-immune sera.

AGM inoculated with PIV5 produce strong antibody responses to both F and HN proteins

One possible explanation for the high dependence of post-immune sera on C’ for neutralization is that the inoculated AGM did not produce antibodies to both F and HN. To determine which viral proteins are recognized by post-immune AGM antibodies, Hep2 cells were mock infected or infected with PIV5 and lysates were analyzed by western blotting. As shown in Fig. 7A, two major viral proteins were specifically recognized by post-immune serum and alignment with known viral proteins indicated these were NP and P protein. No detectable signs of reactivity with SDS-denatured F, HN or M proteins were seen.

Figure 7. PIV5 proteins recognized by antibodies in sera from PIV5-innoculated AGM.

Figure 7

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A) Lysates from Hep2 cells mock infected (M lanes) or infected (I lanes) with PIV5-GFP were analyzed by western blotting using sera from infected AGM animal numbers 1515 and 1536. Control blots that were probed with mono-specific rabbit anti-sera to NP, P or M proteins served as markers for the position of viral proteins. B) CV-1 cells were mock infected or infected at an moi of 10 with PIV5 or with VacV individually expressing PIV5 F, PIV5 HN or control ovalbumin. Cells were fixed and analyzed for surface staining with monkey 1515 post-immune sera.

To determine if post-immune sera recognized F and HN expressed on the cell surface, cells were mock infected or infected with PIV5, or with recombinant vaccinia virus (VacV) expressing either F protein, HN protein or the control protein ovalbumin. Cells were examined for surface staining by post-immune sera followed by FITC-conjugated goat anti-monkey IgG. As seen in Fig. 7B for representative serum from monkey 1515, antibodies recognized PIV5-infected cells but importantly also specifically recognized cells infected with both VacV-F and VacV-HN. Thus, antibodies to both of the two major PIV5 glycoproteins are present in AGM post-immune sera, but their capacity to neutralize virus is highly dependent on intact C’ pathways.

PIV5 derived from AGM cells is refractory to neutralization by NAGS

The above data were generated using PIV5 grown in the bovine cell line MDBK, the standard cell type for generation of PIV5 stocks. To determine if PIV5 grown in AGM versus bovine cells differed in neutralization by non-immune NAGS, PIV5 was grown in the AGM cell line CV1 or MDBK cells and C’-mediated neutralization was carried out. As shown in Fig. 8 for two representative naïve NAGS samples, MDBK-derived virus showed effective C’-dependent neutralization by as high as 1:80 dilution of serum. CV1-derived virus was also neutralized by NAGS, but effective neutralization was not seen at higher dilutions. These data are consistent with the proposal that host cell-derived inhibitors of C’ are incorporated into paramyxovirus particles (Johnson et al., 2012), and that they block C’ pathways in a species specific manner. Most importantly for this study, the finding that AGM-derived PIV5 is more resistant to C’ neutralization by NAGS further highlights the importance of C’ in determining the potency of antibodies that are elicited during an infection by parainfluenza viruses.

Figure 8. PIV5 derived from AGM cells is resistant to C’-mediated neutralization.

Figure 8

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One hundred twenty PFU of PIV5-GFP grown in MDBK or CV-1 cells were incubated for 1 h at 37°C with media alone (control Ctr; left gray bar) or with the indicated dilutions of NAGS (striped bars) or 1:40 of heat inactivated serum (HI, black bars). Remaining infectivity was determined by plaque assays. Results represent the average of four assays, with error bars representing standard deviations. (*) no plaques were detected in these samples.

DISCUSSION

Antibodies can be an important factor in the neutralization of many of the paramyxoviruses associated with pediatric respiratory tract infections (e.g., Delgado and Polack, 2004; Karron and Collins, 2013). However, the contribution of C’ to the potency of the antiviral antibodies that are elicited following a primary parainfluenza virus infection is often overlooked or difficult to analyze. This is in large part due to the very high levels of pre-existing parainfluenza virus antibodies in human sera following repeated exposure at an early age and also due to the use of adult versus infant sera in many analyses of virus neutralization. Here we have used a nonhuman primate model system to determine the contribution of C’ to the neutralizing capacity of antibodies that are elicited following the first exposure to PIV5 infection of the respiratory tract. In the animal population used for this study, we found no evidence for pre-existing PIV5 glycoprotein-specific antibodies, and depletion of antibodies from non-immune serum did not alter C’-mediated neutralization of PIV5. Our most striking finding was that respiratory tract infection elicited strong anti-PIV5 antibodies when assayed by ELISA and these antibodies included responses to both F and to HN, but the neutralizing capacity of these antibodies was highly dependent on intact C’ pathways. Further support for C’-dependence in neutralization by post-immune sera came from the finding that PIV5 virions exhibit strong C1q and IgG deposition when treated with post- but not pre-immune sera. As described below, these results have implications for the potential to harness C’ functions to enhance early immune responses to paramyxovirus infections.

Our finding that anti-PIV5 antibodies have high C’ dependence could reflect the need to generate neutralizing antibodies that are specific for the native form of the parainfluenza virus glycoproteins. It is known that antibodies to both HN and F are elicited by parainfluenza virus infection and they both can contribute to immunity (Karron and Collins, 2013; Spriggs et al., 1987). In the specific case of PIV5, previous work in a hamster model with recombinant VacV expressing F or HN showed that vaccination with F alone generated higher antibody levels than vaccination with HN alone, but anti-HN antibodies were more protective in challenge experiments (Paterson et al, 1987). However, the contribution of C’ to these immune responses and protection was not tested. It is known that there are substantial structural differences between immature and mature forms of the viral glycoproteins (Lamb and Jardetzky, 2007; Mottet et al., 1986). Likewise, point mutations that destabilize the F protein can alter the binding of human antibodies and increase dependence on C’ for neutralization (Johnson et al., 2013). Thus, the inherent conformation of the parainfluenza glycoproteins and epitope accessibility due to virion structures could be a major factor in eliciting antibodies that are initially of low avidity and more dependent on C’ for neutralization. This is consistent with prior proposals that the structure of enveloped virus particles can contribute to differential C’ dependence for neutralization, since antibodies against the VacV extracellular enveloped form are highly dependent on C’ for activity (Benhnia et al., 2009),

Alternatively, the high C’-dependence of antibodies generated in our model system could be a reflection of the particular animals used in our study. The WFU VRC was founded in 1975 with 57 animals imported from the islands of St. Kitts and Nevis (described in Jasinska et al., 2012). Since the mid-1980s, this has been a closed animal breeding colony, with no new animals imported since that time. Previous work on parainfluenza virus infection of AGM (e.g., Schaap-Nutt et al., 2011) showed that animals elicited strong levels of serum antibodies, but the origin of the animals used in these studies was not detailed. Thus, the restricted breeding within a closed colony such as the WFU VRC may contribute to genetic bottlenecks in the ability to generate a range of antibodies that have varying degrees of dependence on C’. The ability to exploit the documented pedigree of the VRC (Jasinska et al., 2012) and the availability of complete genome sequences for many of these animals (http://www.genomequebec.mcgill.ca/compgen/vervet_research/) provides an opportunity to explore the role of genetics in C’ dependence for neutralization.

Our results are based on immune responses elicited following an initial experimental exposure of animals to PIV5, and have not included an analysis of neutralizing capacity following a secondary exposure or boost. For the closely related MuV, human antibodies elicited by early exposure are initially of low affinity and limited isotype diversity, but the antibodies appear to have higher affinity and broader isotype profiles after re-exposure (reviewed in Rubin and Vandermeulen, 2011). Thus, future studies with prime-boost protocols could reveal that high dependence on C’ for neutralization is a reflection of the primary exposure to virus infection. This would be consistent with results with mouse antibodies raised to influenza virus, where C’ effects were primarily seen the case of the antibodies elicited by primary exposure (Feng et al., 2002).

We found that the mechanism of PIV5 neutralization by naïve non-human primate serum was C’-dependent but antibody-independent, and proceeded through formation of massive aggregates, similar to that seen with human sera (Johnson et al., 2008). A similar result was reported for influenza virus, where serum samples from influenza-naïve mice neutralized influenza virus particles by forming viral aggregates, though neither natural IgM nor C’ alone was sufficient for neutralization (Jayasekera et al., 2007). Thus, in contrast to other negative strand RNA viruses such as VSV and HPIV3 which show C’-dependent virion lysis (Johnson et al., 2012; Vasantha et al., 1988), there was no evidence that exposure to AGM serum resulted in release of PIV5 nucleocapsids from within the lipid envelope. Importantly, sera from inoculated animals also neutralized PIV5 through this common mechanism, with the only discernable difference between pre- and post-immune sera being the kinetics of virus aggregation. It is not clear why, despite strong activation of C’, the pathway does not progress to virion lysis. This could reflect failure to progress through the C’ pathway to MAC formation or lack of a functional membrane attack complex (MAC) due to the presence of C’ inhibitors within the virion (Johnson et al., 2013).

Since treatment of PIV5 with post-immune sera does not result in virion lysis, how does C’ enhance the neutralizing potency of monkey anti-PIV5 antibodies? Mehlhop and coworkers (2009) have shown for WNV that C1q was able to lower the threshold number of anti-viral antibodies that are needed for neutralization, and it was proposed that this enhancement was due to changes in epitope accessibility. Thus, C’ factors may alter epitope accessibility of otherwise weakly neutralizing antibodies. Importantly, the contribution of C’ factors to enhanced antibody function in the case of WNV and influenza virus was highly dependent on antibody subclass (Feng et al, 2002; Melhop et al. 2009). This raised the critical point that vaccination strategies designed to induce C’-activating antibodies (e.g., human IgG1 and IgG3) should result in more robust protective responses.

PIV5 is being developed by a number of research groups as a promising vaccine or therapeutic vector (Clark et al, 2011; Chen et al., 2013; Gainey et al., 2008; Thompkins et al., 2007). Consistent with this, AGM inoculated with PIV5-gp160 elicited anti-gp120 IgG and IgM both systemically as well as at the mucosal surface of the respiratory tract. In each of the model systems we have employed - including mice, ferrets and nonhuman primates - inoculation of animals with PIV5 does not result in overt disease symptoms, including no respiratory distress, elevation of temperature, or other signs of disease. It is also noteworthy that animals either previously vaccinated with PIV5 vectors or with natural pre-existing anti-PIV immunity can still respond extremely well to a secondary vaccination with the same PIV5 (Capraro et al., 2008; Chen et al., 2012). Our finding here that anti-viral antibodies elicited by the first respiratory tract exposure to PIV5 are inherently weakly-neutralizing may provide an explanation for this, since the existing antibodies alone may not be able to prevent reinfection.

It is known that human C’ activity is low in neonates and infants when compared to adults. For example, the concentrations of many C’ factors in cord blood and in newborns have been estimated to be only ~10-80% that of adults. Additionally, the functional activity of factors in the neonate C’ cascade is thought to be much lower than can be accounted for by concentration alone (McGreal et al., 2012; Zach and Hostetter, 1989). Given the importance of respiratory tract infections of infants and children (Karron and Collins, 2013), our results highlight the importance of understanding the role of C’ in the initial antibody response of infants to parainfluenza virus natural infections as well as the development of protective vaccines against these and other viruses (Hodgins and Shewen, 2012).

MATERIALS AND METHODS

Cells and viruses

Monolayer cultures of A549, CV-1, MDBK, and Hep2 cells were grown in Dulbecco modified Eagle medium (DMEM) supplemented with 10% HI fetal bovine serum (FBS), penicillin (100 units/ml), streptomycin (100 ug/ml) and 200 mM L-Glutamine. U937 cells were grown in RPMI supplemented as above. Recombinant PIV5 isolates were grown in MDBK cells. HPIV2 was grown and titered in CV-1 cells. Viruses were concentrated by centrifugation through a glycerol cushion (5 h; 25,000 RPM; SW28 rotor), and resuspended virus was further purified by centrifugation on a 30-60% sucrose gradient (2 h; 23,000 RPM; SW28 rotor). The virus band was collected, pelleted again, resuspended in PBS containing 0.75% BSA and stored at -80°C.

WT PIV5 encoding the GFP gene between HN and L (PIV5-GFP) has been described (He et al., 2008). To generate a cDNA encoding PIV5-gp160, the gene encoding the BH8 HIV-1 gp160 protein was removed from plasmid pPE5 (Earl et al., 1990) and altered by PCR to encode 5’ EcoRV and 3’ SalI sites. The resulting DNA fragment was digested with these two enzymes and inserted into the StuI and SalI sites of a modified version of pBH276 (He et al., 1997). Recombinant PIV5 was recovered from transfected cells as described previously (Parks et al., 2002).

Concentration-dependent virus neutralization by normal sera and HI sera was carried out in 200 ul reactions for 1 h at 37°C as previously described (Johnson, et al., 2012), with remaining infectivity determined by plaque assay on CV-1 cells. Results are the average of four to six reactions, with the significance of data points calculated using the student's t-test. Antibodies were depleted from NAGS by treatment of a 1:20 dilution of NAGS with 50 ul of Protein G-sepharose on ice for 15 min. Samples were then used in neutralization assays as described above.

Animal Procedures

All procedures were approved by the Wake Forest University Animal Care and Use Committee. Procedures were based on published approaches for infection of AGM with HPIV2 (Schaap-Nutt et al., 2011). Healthy 3 year-old African Green monkeys (Chlorocebus aethiops) were sedated with ketamine, and placed in a dorsal position before receiving 108 pfu of purified PIV5-gp160 delivered in a total of 2 ml of sterile PBS. One ml was delivered to the trachea and 0.5 ml was delivered to each nostril. Animals were monitored for resting respiration rate, attitude, and signs of respiratory disease. To collect BAL, 15 mls of sterile PBS was administered into the trachea followed by a 15 ml bolus of air. PBS was then aspirated with a syringe, adjusted to 0.5% bovine serum albumin and stored at -80°C. Blood was collected by femoral venipuncture at the days pi indicated in the figure legends and allowed to clot for 1 h at 37°C. The clot was retracted and serum was separated by centrifugation, aliquoted and stored at -80°C. Complement inactivated serum was prepared by heating NAGS at 56°C for 30 min.

ELISA

Anti-PIV5 ELISAs were performed as described previously (Johnson et al. 2008). Briefly, MaxiSorp 96-well ELISA plates (Nunc) were coated with 1 ug of sucrose gradient-purified PIV5. For gp160 ELISAs, plates were coated with 0.2 ug of recombinant gp120 (reagent 11784 supplied by NIH AIDS Research and Reference Program). Plates were incubated at 4°C overnight, washed three times with PBS/Tween (0.2%), and wells were blocked with 200 ul of PBS containing 2% BSA for 2 hr at room temperature. Dilutions of sera were added to wells, incubated for 1 hr, and wells were washed with PBS/Tween before incubation for 1 hr with HRP-conjugated goat anti-monkey IgG or IgM (Jackson ImmunoResearch Laboratories, PA) and development with substrate TMB (tetramethylbenzidine dihydrochloride, Sigma). The absorbance was determined at 450 nm on a Labsystems Multiskan Plus plate reader (Fisher Scientific, GA).

Electron microscopy

To visualized virions, purified PIV5 particles were mixed with NAGS at a dilution of 1:20 and incubated in fluid phase for various time points (0, 15, 30 and 60 mins) at 37°C followed by addition of the samples onto carbon coated 200 mesh gold Formvar carbon support grids (catalog number CF200-AU; Electron Microscopy Sciences, Hartfield, PA). After adsorption for 10 mins, grids were fixed with 2.5% glutaraldehyde, washed with PBS, negatively stained with 2% phosphotungstic acid and observed under Technai transmission electron microscope. Alternatively, virions were treated with NAGS (1:20 dilution), blocked with 1% BSA in PBS and then probed with anti-C1q or anti-human IgG polyclonal antibody at a 1:25 dilution PBS with 1% BSA. C1q deposition was detected with 12 nm gold-labeled donkey anti-goat antibody (Jackson Immunoresearch Laboratories, PA). Binding of human antibody was analyzed using 6 nm gold-labeled goat anti-human secondary antibody.

Western blotting and immunofluorescense

. Western blotting was carried out as described previously using either AGM sera or mono-specific rabbit antisera to the PIV5 P, M and NP proteins (Parks et al., 2002). Alternatively, blots were probed with goat anti-gp120 polyclonal antibody (US Biologicals catalog number H6003-35). Blots were visualized by enhanced chemiluminescence and exposure to film.

Immunofluorescence was carried out as described previously (Manuse and Parks, 2009). Briefly, CV-1 cells infected at an moi of 10 with PIV5, or with recombinant vaccinia viruses expressing the PIV5 F, PIV5 HN (Paterson et al., 1987) or control ovalbumin (Parks and Alexander-Miller, 2002). At 18 h pi, cells were fixed in paraformaldehyde, and stained with a 1:500 dilution of monkey post immune sera followed by a 1:1000 dilution FITC-conjugated goat anti-monkey IgG (Serotec). As a positive control, mouse polyclonal anti-PIV5 serum was generated by intranasal infection of Balb/c mice with PIV5 and collection of sera at day 28 post infection. Images were captured using QImaging digital camera and processed using Q-capture software. Exposure times were manually set to be constant between samples.

  • PIV5 was neutralized by serum from naïve AGM through a Complement-dependent mechanism.

  • The neutralizing capacity of AGM post-immune anti-PIV5 antibodies was dependent on complement.

  • These results highlight the role of complement in antibody responses to parainfluenza viruses.

  • The work has implications for the design of more effective infant vaccine strategies.

ACKNOWLEDGEMENTS

We thank Ken Grant for help with electron microscopy, Matt Jorgensen, Tara Chavanne and Tyler Aycock for expertise in animal work, Reay Paterson for the gift of recombinant vaccinia viruses expressing PIV5 glycoproteins, and acknowledge the excellent technical assistance of Ellen Young. This work was supported by NIH grants AI083253 (GDP) and AI101675 (GDP), and by the flow cytometry core and imaging core of the WFUCCC (NCI CCSG P30CA012197). Animal resources were supported by NIH grant RR019963/OD010965 (Kaplan). AEM is supported by NIH Training Award Grant T32 OD010957.

Footnotes

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