A host-range restricted parainfluenza virus type 3 (PIV3) expressing the human metapneumovirus (hMPV) fusion protein elicits protective immunity in African green monkeys

Abstract

Human metapneumovirus (hMPV) infection causes respiratory tract disease similar to that observed during human respiratory syncytial virus infection (hRSV). hMPV infections have been reported across the entire age spectrum although the most severe disease occurs in young children. No vaccines, chemotherapeutics or antibodies are presently available for preventing or treating hMPV infections. In this study, a bovine/human chimeric parainfluenza virus type 3 (b/h PIV3) expressing the human parainfluenza type 3 (hPIV3) fusion (F) and hemagglutinin-neuraminidase (HN) proteins was engineered to express hMPV fusion (F) protein from the second genome position (b/h PIV3/hMPV F2) with the goal of generating a novel hMPV vaccine. b/h PIV3/hMPV F2 was previously shown to protect hamsters from challenge with wt hMPV (Tang RS, Schickli JH, Macphail M, Fernandes F, Bicha L, Spaete J, et al. Effects of human metapneumovirus and respiratory syncytial virus antigen insertion in two 3' proximal genome positions of bovine/human parainfluenza virus type 3 on virus replication and immunogenicity. J Virol 2003;77:10819-28) and is here further evaluated for efficacy and immunogenicity in African green monkeys (AGMs). AGMs immunized intranasally and intratracheally with b/h PIV3/hMPV F2 generated hMPV- and hPIV3-specific humoral and cellular immune responses and were protected from wt hMPV infection. In a separate study, the host-range restriction of b/h PIV3/hMPV F2 replication relative to wt hPIV3 was performed in rhesus monkeys to demonstrate attenuation. These studies showed that b/h PIV3/hMPV F2 was immunogenic, protective and attenuated in non-human primates and warrants further evaluation in humans as a vaccine candidate for prevention of hMPV-associated respiratory tract diseases.

Fig. 1

Outline of the AGM primate study design from day 21 to day 56. Serum was collected at the days indicated. Three groups of four animals were inoculated intranasally (IN) and intratracheally (IT) on day 1 with wt hMPV/NL/1/00, b/h PIV3/hMPV F2 or placebo. On day 28, AGMs were challenged with wt hMPV/NL/1/00 by intranasal and intratracheal administrations.

Fig. 2

Mean daily shedding of b/h PIV3/hMPV F2, wt hMPV/NL/1/00 and placebo in the URT and LRT of AGMs (n = 4 for each group) are shown for 12 days post-immunization and 10 days post-challenge. Virus titers were determined by plaque assays immunostained with hMPV-specific polyclonal antisera. The limit of detection is 1.3 log₁₀ pfu/ml. (A) The mean daily virus titers shed in the URT of AGMs post-vaccination and (B) in the LRT are shown. Shedding of the challenge virus (wt hMPV/NL/1/00) in the URT (C) and LRT (D) of AGMs immunized with hMPV/NL/1/00, b/h PIV3/hMPV F2 and placebo are shown for 12 days post-challenge in the URT and 9 days in the LRT. hMPV/NL/1/00 (○), b/h PIV3/hMPV F2 (□), placebo (▵).

Fig. 3

Elispot assay using PBMCs from African green monkeys immunized with placebo, b/h PIV3/hMPV F2 or hMPV/NL/1/00 (n = 4 in each group). The frequencies of IFN-γ secreting T cells per 10⁵ input cell stimulated with uninfected Vero cell lysate, wt hMPV/NL/1/00 or wt hPIV3/JS is shown with standard deviations. The number of vaccinated animals in each group is four. Day 1 = pre-vaccination, day 28 = pre-challenge, day 56 = post wt hMPV/NL/1/00 challenge.

Fig. 4

Mean daily shedding of b/h PIV3/hMPV F2, wt hPIV3/JS (Study A) and rbPIV3, b/h PIV3 (Study B) in the URT and LRT of rhesus monkeys (n = 4 in each group). Virus titers were determined by plaque assays immunostained with hMPV or PIV3-specific polyclonal antisera. The limit of detection is 1.3 log₁₀ pfu/ml. (A) The mean daily virus titers shed in the URT of rhesus monkeys post-vaccination and (B) in the LRT are shown. hPIV3/JS (▵), b/h PIV3/hMPV F2 (○), rbPIV3 (●) and b/h PIV3 (▴).

Fig. 5

Mean peak titers of rbPIV3, b/h PIV3, b/h PIV3/hMPV F2 and wt hPIV3/JS shed in the URT (NP) and LRT (BAL) of rhesus monkeys (n = 4 in each group). The limit of detection is 1.3 log₁₀ pfu/ml. Two animals in the b/h PIV3 group did not shed the administered virus in the URT. One animal in b/h PIV3/hMPV F2 group did not shed vaccine virus in both the URT and LRT. The standard error of the mean for each treatment group is shown. The NP and BAL mean peak titers between b/h PIV3/hMPV F2 and hPIV3/JS are statistically different, P = 0.0571 (two-sided Wilcoxon exact P-values) and are indicated by asterisks. Differences between b/h PIV3/hMPV F2 and rbPIV3 or b/h PIV3 are not significant (P > 0.05).