The neutralizing capacity of antibodies elicited by parainfluenza virus infection of African Green Monkeys is dependent on complement

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.

Keywords: Complement; Immune; Parainfluenza; Vaccine.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.