Mucosal parainfluenza virus-vectored vaccine against Ebola virus replicates in the respiratory tract of vector-immune monkeys and is immunogenic

Abstract

We previously used human parainfluenza virus type 3 (HPIV3) as a vector to express the Ebola virus (EBOV) GP glycoprotein. The resulting HPIV3/EboGP vaccine was immunogenic and protective against EBOV challenge in a non-human primate model. However, it remained unclear whether the vaccine would be effective in adults due to preexisting immunity to HPIV3. Here, the immunogenicity of HPIV3/EboGP was compared in HPIV3-naive and HPIV3-immune Rhesus monkeys. After a single dose of HPIV3/EboGP, the titers of EBOV-specific serum ELISA or neutralization antibodies were substantially less in HPIV3-immune animals compared to HPIV3-naive animals. However, after two doses, which were previously determined to be required for complete protection against EBOV challenge, the antibody titers were indistinguishable between the two groups. The vaccine virus appeared to replicate, at a reduced level, in the respiratory tract despite the preexisting immunity. This may reflect the known ability of HPIV3 to re-infect and may also reflect the presence of EBOV GP in the vector virion, which confers resistance to neutralization in vitro by HPIV3-specific antibodies. These data suggest that HPIV3/EboGP will be immunogenic in adults as well as children.

Fig. 1

Design of the study. The arrows above the horizontal lines indicate virus inoculations, and those below indicate days of blood sample collection. Days of virus inoculations and blood samplings are counted after the first dose of HPIV3 (left side) or the first dose of HPIV3/EboGP (right side). The horizontal bars indicate collections of NS and TL on days 2, 4, 6, 8 and 10.

Fig. 2

Immunogenicity of HPIV3/EboGP in HPIV3-immune (grey bars) and HPIV3-naïve (white bars) monkeys. The days on which HPIV3 (A, left side) or HPIV3/EboGP (A, right side, B, C, D) were administered are indicated with arrows. The days after dose 1 of HPIV3 (A, left side) or HPIV3/EboGP (A, right side, B, C, D) are indicated. All titers are expressed as reciprocal mean log₂ values. A. HPIV3-specific serum antibody (Ab) levels determined by HAI. Left side, antibody titers (HPIV3-immune group only) after infection with HPIV3, mean values ± SE for the four animals in the group; right side, antibody titers after infection with HPIV3/EboGP of the two HPIV3-naïve animals, mean values, and the four HPIV3-immune animals, mean values ± SE. B, C, EBOV-specific serum IgG (B) and IgA (C) quantitated by ELISA using inactivated purified EBOV as antigen. Mean titers of the two HPIV3-naïve and mean titers ± SE of the four HPIV3-immune animals are shown. However, for days 28 and 56 (bars indicated with X), the data for the two HPIV3-naïve animals were augmented by analysis, in parallel, of samples from three additional animals from a previous study in which the monkeys were immunized exactly as in the present study (Bukreyev et al., 2007): thus, for these time points, the HPIV3-naïve group is represented by mean values ± SE of a total of five samples. D. EBOV-neutralizing (neut) serum antibodies analyzed using VSVΔG/ZEBOVGP, which is a recombinant VSV bearing an envelope in which EBOV GP is the sole viral surface antigen. Mean titers of the two HPIV3-naïve and mean titers ± SE of the four HPIV3-immune animals are shown. Comparisons of IgG and IgA responses (panels B and C, respectively) between the HPIV3-naïve and the HPIV3-immune monkeys on days 28 and 56 are indicated by horizontal lines; statistically significant differences (p<0.05) are indicated by asterisks. For the samples in which antibodies were not detected, the value 1 log₂ was assigned for calculation of the mean (panels A, C, D).

Fig. 3

Shedding of the HPIV3/EboGP vaccine virus in HPIV3-naïve and HPIV3-immune monkeys. NS and TL samples were collected on the indicated days and analyzed later in parallel by plaque titration or quantitative RT-PCR. Each bar represents an individual monkey.A. Plaque titration of NS and TL specimens after dose 1. The limit of detection was 5 PFU/ml; for the samples in which virus was not detected, values two-fold below the limit of detection were assigned. Virus was not detected in any animal after dose 2 (see Results).B, C. Quantitative RT-PCR analysis of NS and TL specimens after dose 1 (B) and dose 2 (C). The quantitative RT-PCR results are expressed as PFU/ml based on comparison with a standard curve constructed by quantitative RT-PCR analysis of RNA samples from serial dilutions of a preparation of HPIV3/EboGP with a known PFU/ml concentration. The non-specific background signal, determined using NS and TL from monkeys from an unpublished study that were inoculated with a Newcastle disease virus vector, was 2.8–2.9 and 3.0–3.1 log₁₀ PFU equivalents/ml in NS and TL, respectively. The assay was repeated four times.

Fig. 4

Analysis of NS and TL specimens collected after inoculation of HPIV3-naïve monkeys with HPIV3/EboGP or UV-inactivated HPIV3/EboGP for the presence of infectious virus by plaque titration (A) or the presence of HPIV3/EboGP RNA by quantitative RT-PCR (B) calculated as in the assay shown in Fig. 3 (B, C). Each bar represents an individual monkey. In part A, the limit of detection was 5 PFU/ml; for the samples in which no virus was detected, values two-fold below the limit of detection were assigned. In part B, the value 2.9 log₁₀, which is 2-fold below the limit of detection and represents the titer detected in the last (least concentrated) dilution of the HPIV3/EboGP standard curve, was assigned to the samples in which RNA was not detected. The assay was repeated two times.

Fig. 5

Evaluation of the ability of NS (A) and TL (B) fluids collected from HPIV3-naïve and HPIV3-immune monkeys to neutralize HPIV3 wild type virus (black bars) or the HPIV3/EboGP vaccine virus (striped bars) added in vitro at 10³ PFU/ml. The following NS and TL specimens or control were combined with HPIV3 or HPIV3/EboGP in the neutralization test: 1, Control in which phosphate buffered saline was added to the HPIV3 or HPIV3/EboGP preparations; 2, NS and TL specimens collected on day 4 after infection of a rhesus monkey with 2×10⁷ PFU of Newcastle disease virus in a previous unpublished study;3 and 4, NS and TL specimens collected from two different HPIV3-naïve monkeys collected on day 2 after the first dose of HPIV3/EboGP; 5 – 8, NS or TL samples collected from four different HPIV3-immune monkeys collected on day 2 after the first dose of HPIV3/EboGP. All specimens were UV-irradiated before use in order to inactivate shed virus. The added test virus was completely neutralized in the case of HPIV3, NS number 5 (indicated with a single asterisk) and for both HPIV3 and HPIV3/EboGP in the case of TL number 6 (two asterisks). The limit of detection was 5 PFU/ml.