Purification and immunogenicity of hemagglutinin from highly pathogenic avian influenza virus H5N1 expressed in Nicotiana benthamiana

Highly pathogenic avian influenza (HPAI) H5N1 is an ongoing global health concern due to its severe sporadic outbreaks in Asia, Africa and Europe, which poses a potential pandemic threat. The development of safe and cost-effective vaccine candidates for HPAI is considered the best strategy for managing the disease and addressing the pandemic preparedness. The most potential vaccine candidate is the antigenic determinant of influenza A virus, hemagglutinin (HA). The present research was aimed at developing optimized expression in Nicotiana benthamiana and protein purification process for HA from the Malaysian isolate of H5N1 as a vaccine antigen for HPAI H5N1. Expression of HA from the Malaysian isolate of HPAI in N. benthamiana was confirmed, and more soluble protein was expressed as truncated HA, the HA1 domain over the entire ectodomain of HA. Two different purification processes were evaluated for efficiency in terms of purity and yield. Due to the reduced yield, protein degradation and length of the 3-column purification process, the 2-column method was chosen for target purification. Purified HA1 was found immunogenic in mice inducing H5 HA-specific IgG and a hemagglutination inhibition antibody. This paper offers an alternative production system of a vaccine candidate against a locally circulating HPAI, which has a regional significance

A Plant-Produced Pfs25 VLP Malaria Vaccine Candidate Induces Persistent Transmission Blocking Antibodies against Plasmodium falciparum in Immunized Mice

Contains fulltext : 128628.pdf (publisher's version ) (Open Access

A Plant-Produced Pfs230 Vaccine Candidate Blocks Transmission of Plasmodium falciparum▿†

Plasmodium falciparum is transmitted to a new host after completing its sexual cycle within a mosquito. Developing vaccines against the parasite sexual stages is a critical component in the fight against malaria. We are targeting multiple proteins of P. falciparum which are found only on the surfaces of the sexual forms of the parasite and where antibodies against these proteins have been shown to block the progression of the parasite's life cycle in the mosquito and thus block transmission to the next human host. We have successfully produced a region of the Pfs230 antigen in our plant-based transient-expression system and evaluated this vaccine candidate in an animal model. This plant-produced protein, 230CMB, is expressed at approximately 800 mg/kg in fresh whole leaf tissue and is 100% soluble. Administration of 230CMB with >90% purity induces strong immune responses in rabbits with high titers of transmission-blocking antibodies, resulting in a greater than 99% reduction in oocyst counts in the presence of complement, as determined by a standard membrane feeding assay. Our data provide a clear perspective on the clinical development of a Pfs230-based transmission-blocking malaria vaccine

Pfs25-CP VLP particle analysis.

<p>(A) Negative stain transmission electron micrograph of Pfs25-CP VLPs shows highly uniform particles of 19.3±2.4 nm in diameter. (B) Transmission electron micrograph of Pfs25-CP VLPs labeled with anti-Pfs25 and gold-labeled anti-mouse antibodies confirms the presence of Pfs25 on the particles. (C) DLS histogram showing a narrow size distribution for Pfs25-CP VLPs. The average hydrodynamic radius is ∼14 nm with a polydispersity of <15%. (D) Analytical SEC showing a single, major eluting species confirmed by Western blot analysis (not shown) to be Pfs25-CP VLP. The void volume of the SRT 1000 column (range 7.5 MDa –50 kDa) is indicated by (a) molecular weight standards indicated by (b) for thyroglobulin, (c) for BSA and (d) for uracil.</p

Evaluation of TB activity in immunized mouse sera by SMFA: one vaccine dose.

a<p>The proportion (percentage) of mosquitoes infected.</p>b<p>Median number of oocysts per mosquito (range).</p>c<p>% reduction = (mean control oocyst – mean test oocyst) ÷ mean control oocyst)*100.</p>d<p>Control.</p>*<p>CP – coat protein.</p

Pfs25-CP VLP purity and identity.

<p>(A) Deduced amino acid sequence of Pfs25-CP with Pfs25 sequence underlined. Amino acids boxed in the sequence were identified by N-terminal sequencing of the SDS-PAGE bands indicated. A Coomassie stain of an SDS-PAGE gel highlights the Pfs25-CP fusion polypeptide (arrowhead ‘a’) and CP monomer polypeptides (arrowheads ‘b’ & ‘c’). N-terminal sequencing of ‘a’ identified the first 5 amino acids of Pfs25, while sequencing of ‘b & c’ identified residue 26 of AlMV CP. (B) Western blot analysis of Pfs25-CP VLPs with an anti-Pfs25 mAb (left panel) and an anti-AlMV CP polyclonal serum (right panel).</p

Schematic diagram of the ‘launch’ vector.

<p>Following agroinfiltration of plants, the sequence between the left border (LB) and the right border (RB) of the plasmid vector is transferred from <i>Agrobacteria</i> into plant cells where expression of the engineered TMV genome is driven by the Cauliflower mosaic virus (CaMV) 35S promoter. TMV replicase then drives amplification of primary transcript, and Pfs25-CP accumulation is then driven by the TMV CP subgenomic promoter (light blue box). Movement protein (MP) facilitates cell-to-cell transfer of viral sequences and is driven by the MP subgenomic promoter (dark blue box).</p