Recombinant nipah virus vaccines protect pigs against challenge

Abstract

Nipah virus (NiV), of the family Paramyxoviridae, was isolated in 1999 in Malaysia from a human fatality in an outbreak of severe human encephalitis, when human infections were linked to transmission of the virus from pigs. Consequently, a swine vaccine able to abolish virus shedding is of veterinary and human health interest. Canarypox virus-based vaccine vectors carrying the gene for NiV glycoprotein (ALVAC-G) or the fusion protein (ALVAC-F) were used to intramuscularly immunize four pigs per group, either with 10(8) PFU each or in combination. Pigs were boosted 14 days postvaccination and challenged with 2.5 x 10(5) PFU of NiV two weeks later. The combined ALVAC-F/G vaccine induced the highest levels of neutralization antibodies (2,560); despite the low neutralizing antibody levels in the F vaccinees (160), all vaccinated animals appeared to be protected against challenge. Virus was not isolated from the tissues of any of the vaccinated pigs postchallenge, and a real-time reverse transcription (RT)-PCR assay detected only small amounts of viral RNA in several samples. In challenge control pigs, virus was isolated from a number of tissues (10(4.4) PFU/g) or detected by real-time RT-PCR. Vaccination of the ALVAC-F/G vaccinees appeared to stimulate both type 1 and type 2 cytokine responses. Histopathological findings indicated that there was no enhancement of lesions in the vaccinees. No virus shedding was detected in vaccinated animals, in contrast to challenge control pigs, from which virus was isolated from the throat and nose (10(2.9) PFU/ml). Based on the data presented, the combined ALVAC-F/G vaccine appears to be a very promising vaccine candidate for swine.

FIG. 1.

Neutralizing antibody response following vaccination and challenge. Antibody titers in sera collected by serial bleeding of individual animals are plotted separately against the day of serum collection. (Top) Logarithms of neutralizing antibody titers for pigs vaccinated with ALVAC-G, starting at dpv 14. (Middle) Logarithms of neutralizing antibody titers for pigs vaccinated with ALVAC-F, starting at dpv 14. (Bottom) Logarithms of neutralizing antibody titers for pigs vaccinated with a combination of ALVAC-G and ALVAC-F, starting at dpv 14. Statistical analysis was not attempted due to the small numbers of animals per group.

FIG. 2.

Antibody response measured by indirect ELISA using binary ethylenimine-inactivated, sucrose gradient-purified NiV as an antigen. Mean antibody titers for each group of vaccinees are plotted against the day of serum collection. (A) Logarithm of mean antibody titers in sera from pigs vaccinated with ALVAC-G. (B) Logarithm of mean antibody titers in sera from pigs vaccinated with ALVAC-F. (C) Logarithm of mean antibody titers in sera from pigs vaccinated with ALVAC-F/G. Statistical analysis was not attempted due to the small numbers of animals per group.

FIG. 3.

Cells harvested prior to vaccination were divided into several aliquots. Supernatants from untreated (nonstimulated) cells were assayed for baseline levels of cytokines (F₀, G₀, and F/G₀). Cytokines from cells treated with live NiV (stimulated) cells (F_s, G_s, and F/G_s) were assayed for cytokine levels induced by NiV. Cells collected 14 days post-primary vaccination were treated with live NiV. These cells were considered restimulated for the respective specific antigens: F and/or G (F_r, G_r, and F/G_r). They were primarily exposed in vivo to the NiV F and/or G antigens in the canarypox virus context and restimulated in vitro for these antigens in the NiV context. F represents ALVAC-F vaccinees, G represents ALVAC-G vaccinees, and F/G represents ALVAC-F/G vaccinees. (A) Mean IL-10 levels produced by PBMC. IL-10 levels for F₀, G₀, and F/G₀ (below an OD of 0.08) were considered negative and are noted on the graph as zero. (B) Mean TNF-α levels produced by PBMC. (C) Mean IFN-γ levels produced by PBMC. IFN-γ quantities for F₀, G₀, F/G₀, and F/G_s were considered below the detectable levels and are noted on the graph as zero. Statistical analysis was not considered due to the small numbers of animals per group.

FIG. 4.

Histologic scoring data in NiV-infected pigs, which were previously immunized with no vaccine (0) or with vaccine against F, G, or both F and G proteins. Shown are the histologic scores for meningoencephalitis (brain), necrosis of lymphoid organs, necrosis of nasal and tracheal epithelium (airway), and the totals.

FIG. 5.

Histologic lesions in challenge control pigs infected with NiV. (Top) Cerebrum of pig 41, showing lymphocytic meningitis with vasculitis (arrow) and encephalitis (arrowheads). (Middle) Trachea of pig 39, showing epithelial erosion (asterisk) and single-cell necrosis associated with lymphocyte infiltration (arrows). (Bottom) Bronchial lymph node of pig 39, showing syncytial cells (arrows) in a focal area of necrosis, with non-necrotic lymphocytes at the periphery (asterisks).