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. 2017 Apr 28;91(10):e02469-16.
doi: 10.1128/JVI.02469-16. Print 2017 May 15.

Attenuated Human Parainfluenza Virus Type 1 Expressing Ebola Virus Glycoprotein GP Administered Intranasally Is Immunogenic in African Green Monkeys

Matthias Lingemann  1   2 , Xueqiao Liu  1 , Sonja Surman  1 , Bo Liang  1 , Richard Herbert  3 , Ashley D Hackenberg  3 , Ursula J Buchholz  1 , Peter L Collins  1 , Shirin Munir  4
Affiliations

Attenuated Human Parainfluenza Virus Type 1 Expressing Ebola Virus Glycoprotein GP Administered Intranasally Is Immunogenic in African Green Monkeys

Matthias Lingemann et al. J Virol. .

Abstract

The recent 2014-2016 Ebola virus (EBOV) outbreak prompted increased efforts to develop vaccines against EBOV disease. We describe the development and preclinical evaluation of an attenuated recombinant human parainfluenza virus type 1 (rHPIV1) expressing the membrane-anchored form of EBOV glycoprotein GP, as an intranasal (i.n.) EBOV vaccine. GP was codon optimized and expressed either as a full-length protein or as an engineered chimeric form in which its transmembrane and cytoplasmic tail (TMCT) domains were replaced with those of the HPIV1 F protein in an effort to enhance packaging into the vector particle and immunogenicity. GP was inserted either preceding the N gene (pre-N) or between the N and P genes (N-P) of rHPIV1 bearing a stabilized attenuating mutation in the P/C gene (CΔ170). The constructs grew to high titers and efficiently and stably expressed GP. Viruses were attenuated, replicating at low titers over several days, in the respiratory tract of African green monkeys (AGMs). Two doses of candidates expressing GP from the pre-N position elicited higher GP neutralizing serum antibody titers than the N-P viruses, and unmodified GP induced higher levels than its TMCT counterpart. Unmodified EBOV GP was packaged into the HPIV1 particle, and the TMCT modification did not increase packaging or immunogenicity but rather reduced the stability of GP expression during in vivo replication. In conclusion, we identified an attenuated and immunogenic i.n. vaccine candidate expressing GP from the pre-N position. It is expected to be well tolerated in humans and is available for clinical evaluation.IMPORTANCE EBOV hemorrhagic fever is one of the most lethal viral infections and lacks a licensed vaccine. Contact of fluids from infected individuals, including droplets or aerosols, with mucosal surfaces is an important route of EBOV spread during a natural outbreak, and aerosols also might be exploited for intentional virus spread. Therefore, vaccines that protect against mucosal as well as systemic inoculation are needed. We evaluated a version of human parainfluenza virus type 1 (HPIV1) bearing a stabilized attenuating mutation in the P/C gene (CΔ170) as an intranasal vaccine vector to express the EBOV glycoprotein GP. We evaluated expression from two different genome positions (pre-N and N-P) and investigated the use of vector packaging signals. African green monkeys immunized with two doses of the vector expressing GP from the pre-N position developed high titers of GP neutralizing serum antibodies. The attenuated vaccine candidate is expected to be safe and immunogenic and is available for clinical development.

Keywords: Ebola GP; Ebola glycoprotein GP; Ebola virus; human parainfluenza virus; human parainfluenza virus type 1; intranasal vaccine; live attenuated vaccine; mucosal vaccine; vaccine; vectored vaccine.

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Figures

FIG 1
FIG 1
Construction of antigenomic cDNAs of rHPIV1-CΔ170 expressing full-length EBOV GP or chimeric EBOV GP with HPIV1 F transmembrane (TM) and cytoplasmic tail (CT) domains from the pre-N (A) or N-P (B) position. The EBOV GP ORF (strain Mayinga; GenBank accession number AF086833.2) was modified by the insertion of a single A residue at the editing site so as to encode full-length GP. The ORF was codon optimized and inserted, as either the full-length GP or as a chimeric form in which the TMCT domain was replaced with that of HPIV1 F, at the pre-N (A) or N-P (B) position of rHPIV1-CΔ170 bearing an attenuating mutation in the P/C ORF (indicated by an asterisk) using the previously introduced (17) unique MluI or AscI restriction sites, respectively. The GP insert was engineered with flanking HPIV1 gene transcription signals to allow for its expression as a separate mRNA. The amino acid alignment of EBOV GP, HPIV1 F, and the chimeric EBOV GP-TMCT, where the TM and CT of EBOV GP has been replaced with that of HPIV1 F (bold), is shown in panel C.
FIG 2
FIG 2
Multistep growth kinetics. Vero cells were infected in triplicate with an MOI of 0.01 TCID50/cell with each indicated HPIV1-CΔ170 virus expressing EBOV GP or GP-TMCT virus. The cells were incubated at 32°C, and aliquots of culture supernatant were collected at 24-h intervals over 6 days. Virus titers were determined by limiting dilution on LLC-MK2 cells using a hemadsorption assay. Titers are reported as log10 TCID50 per milliliter. Mean titers are shown, with the standard deviations as vertical error bars. The statistical significance of the difference between EBOV GP viruses (GP1, GP-TMCT1, GP2, and GP-TMCT2) and the rHPIV1-CΔ170 empty backbone was determined by two-way analysis of variance with Tukey's multiple-comparison posttest and is shown in the table below the graph.
FIG 3
FIG 3
Stability of EBOV GP expression by HPIV1 vectors. (A) Double-staining plaque assay. Vero cells were inoculated with a 10-fold serial dilution of each virus in duplicate, and infected monolayers were incubated for 6 days at 32°C under 0.8% methylcellulose overlay. Immunostaining was performed with mouse monoclonal anti-EBOV GP and goat polyclonal anti-HPIV1 antibodies. Secondary antibodies included infrared dye-labeled donkey anti-mouse 800CW and donkey anti-goat 680LT. Plaque images were acquired with an Odyssey infrared imaging system, and the percentage of plaques expressing EBOV GP was determined. The infrared dyes were pseudocolored to appear red and green for HPIV1 and EBOV GP, respectively. On merging the colors, the plaques coexpressing GP and HPIV1 antigens appeared yellow and those not expressing GP were red. Representative monolayers are shown as examples. (B) Double-staining flow cytometry assay. Vero cells infected with the vectors were stained with anti-HPIV1 F and anti-EBOV GP (KZ52) monoclonal antibodies as described in Materials and Methods and analyzed on a BD FACSCanto II. An example of EBOV GP staining of HPIV1 F+ single live cells is shown as scatter plots, and the stability of EBOV GP expression (percentage of EBOV GP+ cells in HPIV1 F+ population) for all vectors is shown as a table.
FIG 4
FIG 4
Western blot analysis of infected cell lysates and sucrose gradient-purified virions. (A) Cell lysates. Vero cells were infected at an MOI of 3 TCID50/cell with the indicated viruses and incubated for 48 h at 32°C, after which cells were lysed in LDS sample buffer and analyzed by Western blotting. EBOV GP was detected with mouse anti-GP and anti-mouse IRDye 680 RD antibodies. Individual HPIV1 proteins were detected with rabbit polyclonal hyperimmune sera raised individually against peptides derived from HPIV1 N, P, F, and HN proteins, as previously described (17). Tubulin was detected on all blots, as a loading control, using mouse antitubulin and anti-mouse IRDye 680RD antibodies; a representative blot is shown. (B) Sucrose-purified virus particles. LLC-MK2 cells were infected at an MOI of 0.1 TCID50 per cell with the indicated viruses and incubated for 6 days at 32°C, and virus particles were purified by sucrose step gradient centrifugation. For each virus, 1 μg of total protein, measured by BCA assay, was analyzed by Western blotting using the same antibodies as described above. (C) Relative quantification of EBOV GP incorporation into HPIV1 virions. EBOV GP signals on the blots were quantified (Licor-Image Studio) for three independent experiments and normalized to HPIV1 P, which was detected simultaneously on each blot. Quantification is shown relative to GP packaged in GP1 particles (set at a value of 1.0). The statistical significance of the differences in the amount of virion-packaged GP was determined by one-way analysis of variance with Tukey's multiple-comparison posttest and is indicated by four asterisks (P < 0.0001).
FIG 5
FIG 5
Replication of rHPIV1 expressing EBOV GP in AGMs. Groups of four animals were inoculated with 106 TCID50 in 1 ml each via the intranasal and intratracheal routes with the indicated HPIV1-CΔ170 viruses expressing EBOV GP or GP-TMCT. Two animals were inoculated with wt HPIV1 or HPIV3/EboGP. Nasopharyngeal swabs were collected daily (A) and tracheal lavages were performed every other day (B) during the 2 weeks postinoculation. Virus titers were determined by limiting dilution on LLC-MK2 cells and hemadsorption. The average titers are shown as log10 TCID50 per milliliter, with the standard errors of means indicated by error bars. Note that values indicate virus titers after the first dose; replication of the booster dose could not be detected and is not shown.
FIG 6
FIG 6
EBOV GP-specific serum IgG titer. Sera of the immunized AGMs collected at 28 days (d), 35 days (7 days postboost), and 56 days (28 days postboost) after the first immunization were analyzed by an EBOV-GP capture ELISA specific for IgG. Titers for individual animals are shown by symbols. Open circles indicate animals that did not receive the booster dose on day 28. Horizontal bars indicate mean titers; for day 28 p.i., standard deviations are shown as error bars. Values are reported as reciprocal titers of an OD450 of 1.0.
FIG 7
FIG 7
EBOV GP-specific 60% plaque reduction neutralization assay. Serum samples were analyzed for 60% plaque reduction neutralization test (PRNT60) titer using rHPIV3/NotI ΔF-HN/EboGP virus. (A) KZ52, a human monoclonal EBOV GP antibody known to have GP neutralizing activity, was used as a positive control. Rabbit hyperimmune sera against HPIV1 and HPIV3 were included as controls to determine any neutralizing activity of HPIV1- or HPIV3-specific antibodies for rHPIV3/NotI ΔF-HN/EboGP. (B) Sera from the immunized African green monkeys at 28 days, 35 days (7 days postboost), and 56 days (28 days postboost) after the first immunization were analyzed by virus neutralization assay, and the PRNT60 titers were determined for rHPIV3/NotI ΔF-HN/EboGP in the presence of guinea pig complement. Open circles indicate animals that did not receive a booster on day 28 after primary immunization. The average PRNT60 titer for each group is shown with the horizontal bars.
FIG 8
FIG 8
Representative results of the double-staining plaque assay used to determine the stability of EBOV GP expression during replication in African green monkeys. Representative examples of double-stained Vero monolayers are shown from the experiment in Table 1 (see Table 1 for details). (A) wt HPIV1, animal 32956, TL, day P2; (B) GP1, animal 7856, NP, day P1; (C) GP-TMCT1, animal 8392, NP, day P3; (D) GP2, animal 62403, NP, P2; (E) GP-TMCT2, animal 8232, NP, day P2. Vero cells were infected with serially diluted NP or TL samples, incubated for 6 days under a methylcellulose overlay, fixed, and subjected to immunostaining against EBOV GP (green) and HPIV1 (red). A representative plate is shown for each indicated specimen. Plaques that stained for both HPIV1 and EBOV GP appeared yellow; plaques in which expression of EBOV GP was lost appeared red. P2 is the day of peak shedding for that particular animal, P1 is the day before the peak of shedding, and P3 is the day following peak shedding. The complete experiment is summarized in Table 1.
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