Vaccine potential of Nipah virus-like particles

Abstract

Nipah virus (NiV) was first recognized in 1998 in a zoonotic disease outbreak associated with highly lethal febrile encephalitis in humans and a predominantly respiratory disease in pigs. Periodic deadly outbreaks, documentation of person-to-person transmission, and the potential of this virus as an agent of agroterror reinforce the need for effective means of therapy and prevention. In this report, we describe the vaccine potential of NiV virus-like particles (NiV VLPs) composed of three NiV proteins G, F and M. Co-expression of these proteins under optimized conditions resulted in quantifiable amounts of VLPs with many virus-like/vaccine desirable properties including some not previously described for VLPs of any paramyxovirus: The particles were fusogenic, inducing syncytia formation; PCR array analysis showed NiV VLP-induced activation of innate immune defense pathways; the surface structure of NiV VLPs imaged by cryoelectron microscopy was dense, ordered, and repetitive, and consistent with similarly derived structure of paramyxovirus measles virus. The VLPs were composed of all the three viral proteins as designed, and their intracellular processing also appeared similar to NiV virions. The size, morphology and surface composition of the VLPs were consistent with the parental virus, and importantly, they retained their antigenic potential. Finally, these particles, formulated without adjuvant, were able to induce neutralizing antibody response in Balb/c mice. These findings indicate vaccine potential of these particles and will be the basis for undertaking future protective efficacy studies in animal models of NiV disease.

Figure 1. The amount of M, F and G plasmids used at transfection has a bearing on the level of particle formation based on minigenome-encoded reporter gene levels in VLP-infected cells.

Cells were transfected with increasing concentrations of M, or F or G expression plasmids (indicated by a triangle) while keeping the concentration of the other two plasmids fixed. They were co-transfected with the previously optimized minigenome and N, P and L constructs , . Forty eight hours post transfection, the cell SUPs were clarified by centrifugation, and same volume of SUP from each sample was used to infect new cell monolayers (VLP infected) which were transfected 24 hours previously with the core plasmids N, P and L to support replication of the VLP-incorporated minigenome RNA; the VLP-infected cells were harvested 48 hours later for reporter gene analysis. Panel A shows the plasmids transfected in each reaction. Panel B: shows minigenome-encoded CAT activity in VLP infected cell monolayers. Lane 1 is a negative control. Absence of CAT activity in duplicate lanes 2 and 3 indicates that VLP formation, and consequently VLP-incorporated minigenome transfer and expression, does not occur in the absence of M, F and G proteins. The results in lanes 4 through 15 shows that CAT levels varied in VLP infected samples depending on the concentration of M, F and G constructs used at transfection. Thus, the amount of M, F and G plasmids used at transfection had a bearing on the level of particle formation, and the consequent CAT reporter gene transfer and expression. CAT activity in the VLP infected reactions appeared optimal in the boxed lanes 7 and 8. Further analysis of CAT levels in the linear range (data not shown) demonstrated that optimal VLP formation was achieved with the ratios of M, F and G expression plasmids of 3∶1∶1 (lane 7).

Figure 2. Co-expression of NiV proteins G, F and M results in the formation substantial quantities of VLPs morphologically resembling NiV virions.

VLPs released in the transfected cell-supernatant were harvested and purified as described under Methods, and viewed by EM and cryoEM to evaluate their morphology. Under optimized conditions, substantial amounts of VLPs were produced, (A) shows VLP-containing band in the sucrose gradient. Negatively stained sample in (B) show numerous well preserved VLPs. Selected VLPs which were magnified (C) to show clearly the spikes of the glycoproteins present on the VLP surface; an occasional particle had what appeared to be a double fringe (shown with an arrow), a feature normally thought to be associated with Hendra virus particles . (D) Shows cryoelectron micrograph of one of our VLPs. The glycoprotein spikes and their spatial arrangement are seen here even more clearly. (E) Shows functional assembly and immunoreactivity of NiV glycoproteins at the VLP surface. Unfixed particles were stained by immunogold labeling technique using NiV-specific polyclonal antibody and gold labeled secondary antibody. Unfixed particles were used so that only the surface proteins would be available for immunoreactivity. The Figure shows two VLPs with gold-decorated proteins on the VLP surface.

Figure 3. VLP-incorporated NiV proteins.

The Figure shows western blot analysis of NiV VLPs to verify their composition. The VLPs were processed and analyzed by SDS-PAGE as described using manufacturer's instructions. VLP protein bands corresponding in size to NiV proteins G, F₀, F₁ and M were clearly visible.

Figure 4. NiV VLP-induced syncytia in 293 cells is blocked by prior treatment with NiV-specific antibody.

NiV VLPs were pre-incubated for one hour at 37°C with either NiV specific antibody or Junin virus-(JV) specific antibody, or with OPTI-MEM I medium only (untreated VLPs) before inoculating onto 293 cell monolayers grown overnight in 60 mm dishes. The plates were incubated overnight at 37°C and stained with crystal violet. The results show VLP-mediated formation of syncytia (a and b) that were blocked (c) when the VLPs were pretreated with NiV-specific antiserum but not blocked (d) when the VLPs were pre-treated with Junin virus-specific antibody. Images e and f show uninfected 293 cells. Arrow points to syncytia.

Figure 5. NiV VLP-induced immune response in Balb/c mice.

NiV-specific antibody levels of serum samples from mice immunized subcutaneously three times were measured by IFA and by Bio-Plex microsphere methods. Neutralizing antibody response was evaluated by PRNT₅₀. The experiments were done in duplicate. A: For evaluation by IFA, sera from each treatment group were pooled for analysis. The results show serocoversion for each of the four treatment groups. In general, the titers increased progressively with time and with the VLP dose although by day 35, similar titers were seen with the three higher VLP doses. B: Shows neutralizing antibody titers (PRNT₅₀) in sera from each mouse collected on the stated days. Neutralizing antibodies were seen starting on day 28 after primary inoculation. The response was again clearly dose dependent; all mice in the two highest treatment groups (C and D) showed neutralizing response by day 35. Such response was seen in 3 of 5 and 1 of 5 mice in the two lower (B and A respectively) treatment groups.

Figure 6. VLP-induced modulation in transcription profile of genes involved in signaling of innate immune response in 293 cells by PCR Array.

293 cells were grown overnight in 60 mm dishes and were infected with 10 µg of purified VLPs suspended in OPTI-MEM (Invitrogen). Mock infected cells served as negative control. The inoculum was adsorbed on the cell monolayers for 3 hours at 37°C when it was supplemented with fresh OPTI-MEM and further incubated overnight when total cell RNA was extracted according to the manufacturer's (SA Biosciences) instructions. A: shows the integrity of the RNA used for this analysis. Note the ∼2∶1 ratio of 28S:18S which is a good indication of the integrity of the RNA. Equal concentration of the RNA from the mock and VLP-exposed cells was used for expression profiling by RT² PCR Profiler PCR Array according the manufacturer's (SABiosciences) instructions. B: Shows the heat map, it is a visual illustration of the relative expression levels in the VLP-stimulated vs. the “mock” stimulated control cells of the all the genes in the array: The four genes differentially expressed by a factor of ∼4 fold or greater (shown as red squares) are listed below the heat map.