Recombinant newcastle disease virus expressing a foreign viral antigen is attenuated and highly immunogenic in primates

Abstract

Paramyxoviruses such as human parainfluenza viruses that bear inserts encoding protective antigens of heterologous viruses can induce an effective immunity against the heterologous viruses in experimental animals. However, vectors based on common human pathogens would be expected to be restricted in replication in the adult human population due to high seroprevalence, an effect that would reduce vector immunogenicity. To address this issue, we evaluated Newcastle disease virus (NDV), an avian paramyxovirus that is serotypically distinct from common human pathogens, as a vaccine vector. Two strains were evaluated: the attenuated vaccine strain LaSota (NDV-LS) that replicates mostly in the chicken respiratory tract and the Beaudette C (NDV-BC) strain of intermediate virulence that produces mild systemic infection in chickens. A recombinant version of each virus was modified by the insertion, between the P and M genes, of a gene cassette encoding the human parainfluenza virus type 3 (HPIV3) hemagglutinin-neuraminidase (HN) protein, a test antigen with considerable historic data. The recombinant viruses were administered to African green monkeys (NDV-BC and NDV-LS) and rhesus monkeys (NDV-BC only) by combined intranasal and intratracheal routes at a dose of 10(6.5) PFU per site, with a second equivalent dose administered 28 days later. Little or no virus shedding was detected in nose-throat swabs or tracheal lavages following immunization with either strain. In a separate experiment, direct examination of lung tissue confirmed a highly attenuated, restricted pattern of replication by parental NDV-BC. The serum antibody response to the foreign HN protein induced by the first immunization with either NDV vector was somewhat less than that observed following a wild-type HPIV3 infection; however, the titer following the second dose exceeded that observed with HPIV3 infection, even though HPIV3 replicates much more efficiently than NDV in these animals. NDV appears to be a promising vector for the development of vaccines for humans; one application would be in controlling localized outbreaks of emerging pathogens.

FIG. 1.

Genome map of recombinant NDV-BC bearing an insert encoding the HN glycoprotein of HPIV3; the insertion strategy and adjoining sequences were identical for NDV-LS and are not shown. The parental NDV-BC genome is shown at the bottom with the engineered XbaI site indicated. The NDV genes are shown as black rectangles; Le and Tr are the extragenic leader and trailer regions. The NDV-BC/HN vector is shown above the parent with the HPIV3 HN insert as a cross-hatched horizontal bar. The nucleotide sequence flanking the HN ORF is shown above in the expanded view in the positive sense: the sequence of the DNA that was inserted into the XbaI site is bracketed, gene start and gene end transcription signals are boxed, intergenic nucleotides are indicated, the ATG and TAA initiation and termination signals of the HN ORF are underlined, and the remainder of the HN ORF is deleted and is indicated by three dots.

FIG. 2.

Northern blot analysis of the transcription of the HPIV3 HN mRNA by NDV-BC/HN and NDV-LS/HN in vitro. (A) DF-1 chicken cells (lanes 1 to 4) or LLC-MK-2 rhesus monkey cells (lanes 5 and 6) were infected with NDV-BC (lane 1), NDV-BC/HN (lane 2), NDV-LS (lane 3), NDV-LS/HN (lane 4), and HPIV3 (lane 5) or were mock infected (lane 6). (B) African green monkey Vero cells were infected with NDV-BC (lane 1), NDV-BC/HN (lane 2), NDV-LS (lane 3), NDV-LS/HN (lane 4), and HPIV3 (lane 5) or were mock infected (lane 6). Cells were infected at an MOI of 2 PFU (NDV recombinant viruses) or 2 TCID₅₀ (HPIV3) per cell. Cells were harvested 24 h postinfection, and total intracellular RNA was harvested, separated on a formaldehyde agarose gel, and analyzed by Northern blot hybridization with a dsDNA probe to the HPIV3 HN gene. The positions of the HPIV3 HN mRNA and a readthrough mRNA are indicated.

FIG. 3.

Western blot analysis of the expression of the HPIV3 HN protein by NDV-BC/HN in vitro and the incorporation of the HPIV3 HN protein into the NDV virus particle. (A) Intracellular expression. DF-1 cells (lanes 2 and 3) or LLC-MK2 cells (lanes 4 and 5) were infected at an MOI of 2 PFU per cell with NDV-BC (lane 2) or NDV-BC/HN (lane 3) or with 2 TCID₅₀ per cell of HPIV3 (lane 4) or were mock infected (lane 5). The cells were harvested 24 h postinfection, separated by electrophoresis on a 4 to 12% gel under denaturing and reducing conditions, and subjected to Western blot analysis with a rabbit antiserum raised against gradient-purified HPIV3. Lane 1 contains molecular weight markers with molecular weights indicated to the left. Some of the major HPIV3 proteins are indicated at the right. (B) Incorporation of the HPIV3 HN protein into NDV particles. Preparations of NDV-BC (lane 2), NDV-BC/HN (lane 3), and HPIV3 (lane 4) were partially purified by sedimentation in sucrose gradients and were subjected to gel electrophoresis and Western blot analysis as described in the legend to panel A using HPIV3-specific antiserum. Lane 1 contains markers whose molecular weights are shown at the left. Some of the major HPIV3 proteins are indicated at the right.