ROLE OF FUSION PROTEIN IN NEWCASTLE DISEASE VIRUS PATHOGENESIS

The fusion (F) protein of Newcastle disease virus (NDV) is a type I membrane glycoprotein that mediates the merger of the viral envelope to the host cell membrane. The F protein activation initiates a series of conformational changes in the F protein leading to membrane merge which occurs at the cell surface at neutral pH thus modulating NDV entry and spread. The present studies have given an insight to understand the role of F protein in NDV pathogenesis by using established reverse genetic techniques. The F gene of NDV has six glycosylation sites, two of which are present in heptad repeats that facilitate conformational changes during fusion process. To understand the importance of the glycosylation sites in NDV replication and virulence, each site was eliminated individually and in combination on a cDNA clone of NDV strain BC. Our results suggest that glycosylation of F protein plays a major role in virulence and some of the N-glycosylation sites are critical for fusogenicity of the F protein thus modulating NDV infectivity. The F protein is synthesized as an inactive precursor, F0, which is only fusogenic after cleavage into disulfide-linked F1 and F2 polypeptides by host cell proteases. The amino acid sequence surrounding the F protein cleavage site determines the virulence of NDV, since different host proteases that cleave the F protein of virulent strains are present in more tissues than those that cleave the F protein of non-virulent strains. The role of conserved glutamine residue in NDV F protein cleavage site in viral pathogenesis has been examined. This study has helped us to understand the requirement of F protein cleavage site conserved amino acids in proteolytic processing and viral infectivity. Further in this study, the role of F protein cytoplasmic domain and conserved cysteine residues in viral pathogenesis have been explored using reverse genetics. These regions have been suggested to play important roles in F protein conformation, stability and thus affecting the fusion process and viral infectivity. In summary, the purpose of this work is to determine the important domains and residues of the NDV F protein that facilitates fusion process and regulates viral pathogenesis and immunogenicity. An understanding of how NDV F protein fusion process are regulated may lead to the creation of more effective therapies and better vaccine against NDV and other paramyxoviruses in general

Comparative immunogenicity of HIV-1 gp160, gp140 and gp120 expressed by live attenuated newcastle disease virus vector.

The development of a vaccine against human immunodeficiency virus-1 (HIV-1) capable of inducing broad humoral and cellular responses at both the systemic and mucosal levels will be critical for combating the global AIDS epidemic. We previously demonstrated the ability of Newcastle disease virus (NDV) as a vaccine vector to express oligomeric Env protein gp160 and induce potent humoral and mucosal immune responses. In the present study, we used NDV vaccine strain LaSota as a vector to compare the biochemical and immunogenic properties of vector-expressed gp160, gp120, and two versions of gp140 (a derivative of gp160 made by deleting the transmembrane and cytoplasmic domains), namely: gp140L, which contained the complete membrane-proximal external region (MPER), and gp140S, which lacks the distal half of MPER. We show that, similar to gp160, NDV-expressed gp140S and gp120, but not gp140L, formed higher-order oligomers that retained recognition by conformationally sensitive monoclonal antibodies. Immunization of guinea pigs by the intranasal route with rLaSota/gp140S resulted in significantly greater systemic and mucosal antibody responses compared to the other recombinants. Immunization with rLaSota/140S, rLaSota/140L rLaSota/120 resulted in mixed Th1/Th2 immune responses as compared to Th1-biased immune responses induced by rLaSota/160. Importantly, rLaSota/gp140S induced neutralizing antibody responses to homologous HIV-1 strain BaL.26 and laboratory adapted HIV-1 strain MN.3 that were stronger than those elicited by the other NDV recombinants. Additionally, rLaSota/gp140S induced greater CD4+ and CD8+ T-cell responses in mice. These studies illustrate that rLaSota/gp140S is a promising vaccine candidate to elicit potent mucosal, humoral and cellular immune responses to the HIV-1 Env protein

Evaluation of the replication, pathogenicity, and immunogenicity of avian paramyxovirus (APMV) serotypes 2, 3, 4, 5, 7, and 9 in rhesus macaques.

Avian paramyxoviruses (APMV) serotypes 1-9 are frequently isolated from domestic and wild birds worldwide. APMV-1 (also called Newcastle disease virus, NDV) is attenuated in non-human primates and is being developed as a candidate human vaccine vector. The vector potential of the other serotypes was unknown. In the present study, we evaluated nine different biologically- or recombinantly-derived APMV strains for the ability to replicate and cause disease in rhesus macaque model. Five of the viruses were: biologically-derived wild type (wt) APMV-2, -3, -5, -7 and -9. Another virus was a recombinant (r) version of wt APMV-4. The remaining three viruses were versions of wt rAPMV-2, -4 and -7 in which the F cleavage site had been modified to be multi-basic. Rhesus macaques were inoculated intranasally and intratracheally and monitored for clinical disease, virus shedding from the upper and lower respiratory tract, and seroconversion. Virus shedding was not detected for wt APMV-5. Very limited shedding was detected for wt rAPMV-4 and modified rAPMV-4, and only in a subset of animals. Shedding by the other viruses was detected in every infected animal, and usually from both the upper and lower respiratory tract. In particular, shedding over a number of days in every animal was observed for modified rAPMV-2, wt APMV-7, and modified rAPMV-7. Modification of the F protein cleavage site appeared to increase shedding by wt rAPMV-2 and marginally by wt rAPMV-4. All APMVs except wt APMV-5 induced a virus-specific serum antibody response in all infected animals. None of the animals exhibited any clinical disease signs. These results indicate that APMVs 2, 3, 4, 7, and 9 are competent to infect non-human primates, but are moderately-to-highly restricted, depending on the serotype. This suggests that they are not likely to significantly infect primates in nature, and represent promising attenuated candidates for vector development

Detection of NDV-expressed Env proteins present in infected-cell lysates (A) and the cell culture medium (B).

<p>DF1 cells were infected with indicated viruses at an MOI of 0.01 PFU. After 48 h, the cell culture medium supernatants and cells were collected and processed. (A) The cell lysates were prepared from cells and subjected to SDS-PAGE under reducing conditions. (B) The cell culture medium supernatants were concentrated 10x by passing through Amicon filters and subjected to SDS-PAGE under reducing conditions. The gels were analyzed by Western blotting using a pool of gp120-specific monoclonal antibodies. The positions of HIV-1 gp160, gp140 precursor gp120 are indicated by arrows in the right margin. Molecular masses of marker proteins (in kilodaltons) are shown in the left margin.</p

HIV-1 Env-specific CD4+ (panel A) and CD8+ (panel B) T cell response.

<p>Mice in groups of 6 were immunized with 10⁵ PFU/ml of the indicated rNDV by the i.n. route on days 0 and 14. On day 56, splenocytes were isolated, stimulated with a pool of overlapping Env peptides, and processed for intracellular cytokine staining for IFN-γ and CD4 and CD8.</p

Oligomeric status of NDV-expressed Env proteins.

<p>DF1 cells were infected with indicated viruses at an MOI of 0.01 PFU. After 48 h, the cells were collected and cross-linked with DSP and subjected to SDS-PAGE under reducing (+R) or non-reducing (-R) conditions. The gels were analyzed by Western blotting using a pool of gp120-specific monoclonal antibodies. The positions of HIV-1 gp160, gp140 precursor gp120 are indicated by arrows in the right margin. Molecular masses of marker proteins (in kilodaltons) are shown in the left margin.</p

NDV-specific total IgG (panel A), and HIV-1 gp120-specific total IgG (panel B), IgG1 (panel C) and IgG2 (panel D) responses in guinea pig sera.

<p>The guinea pigs were immunized with the indicated rNDVs by the i.n. route. (A) The guinea pig sera were analyzed for NDV-specific antibodies by commercial NDV ELISA kits (Synbiotics Corporation). Mean ELISA end-point titers of NDV-specific serum antibodies on days 28 and 56 are shown. (B-D) The guine pig sera were analyzed by HIV-1 gp120 specific total IgG, IgG1 and IgG2 antibodies by isotype-specific ELISA with purified gp120. Mean ELISA end-point titers of gp120-binding serum antibodies of the indicated isotype on days 0, 7, 14, 21, 28, 42, 56, 70 and 90 are shown. Antibodies specific to gp120 were not detected in any animal on any day in the control rLaSota group. The graph shows the geometric mean value ± SEM for 3 animals in rLaSota, rLaSota/gp160 and rLaSota/gp140L groups, 6 animals in rLaSota/gp140S group and 5 animals in rLaSota/gp120 group. Arrows indicate time of rNDV immunizations on days 0 and 14. Statistical differences between the groups were calculated by unpaired t test (two-tailed). 1* indicates statistically significant differences (<i>P</i><0.05) of rLaSota/gp140S vs. rLaSota/gp160, rLaSota/gp140L and rLaSota/gp120 groups. 2* indicates statistically significant differences (<i>P</i><0.05) of rLaSota/gp140S vs. rLaSota/gp160 and rLaSota/gp140L groups.</p