Influenza nucleoprotein delivered with aluminium salts protects mice from an influenza virus that expresses an altered nucleoprotein sequence

Influenza virus poses a difficult challenge for protective immunity. This virus is adept at altering its surface proteins, the proteins that are the targets of neutralizing antibody. Consequently, each year a new vaccine must be developed to combat the current recirculating strains. A universal influenza vaccine that primes specific memory cells that recognise conserved parts of the virus could prove to be effective against both annual influenza variants and newly emergent potentially pandemic strains. Such a vaccine will have to contain a safe and effective adjuvant that can be used in individuals of all ages. We examine protection from viral challenge in mice vaccinated with the nucleoprotein from the PR8 strain of influenza A, a protein that is highly conserved across viral subtypes. Vaccination with nucleoprotein delivered with a universally used and safe adjuvant, composed of insoluble aluminium salts, provides protection against viruses that either express the same or an altered version of nucleoprotein. This protection correlated with the presence of nucleoprotein specific CD8 T cells in the lungs of infected animals at early time points after infection. In contrast, immunization with NP delivered with alum and the detoxified LPS adjuvant, monophosphoryl lipid A, provided some protection to the homologous viral strain but no protection against infection by influenza expressing a variant nucleoprotein. Together, these data point towards a vaccine solution for all influenza A subtypes

Novel Platforms for the Development of a Universal influenza vaccine

Despite advancements in immunotherapeutic approaches, influenza continues to cause severe illness, particularly among immunocompromised individuals, young children, and elderly adults. Vaccination is the most effective way to reduce rates of morbidity and mortality caused by influenza viruses. Frequent genetic shift and drift among influenzavirus strains with the resultant disparity between circulating and vaccine virus strains limits the effectiveness of the available conventional influenza vaccines. One approach to overcome this limitation is to develop a universal influenza vaccine that could provide protection against all subtypes of influenza viruses. Moreover, the development of a novel or improved universal influenza vaccines may be greatly facilitated by new technologies including virus-like particles, T-cell-inducing peptides and recombinant proteins, synthetic viruses, broadly neutralizing antibodies, and nucleic acid-based vaccines. This review discusses recent scientific advances in the development of next-generation universal influenza vaccines.Funding Agencies|GlaxoSmithKline Biologicals SA; Marie-Curie IEF grant SAMUFLU FP7-PEOPLE-IEF [626283]; Marie-Curie ITN grant HOMIN FP7-PEOPLE-ITN [626283]</p

Cell-penetrating porcine single-chain antibodies (transbodies) to nonstructural protein 1β (NSP1β) of PRRSV inhibit the virus replication

Abstract Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), causes porcine reproductive and respiratory syndrome (PRRS) worldwide, especially among domestic pigs with enormous economic impact for the pig industry. Current vaccines confer limited effectiveness while no direct-acting anti-PRRS is available. Non-structural protein (NSP) 1β, a cysteine-like protease (CLPro) of PRRSV is pivotal for viral polyprotein processing, subgenomic RNA synthesis and evasion of host innate immunity. Therefore, agent that interferes with the NSP1β bioactivities should lead to the virus replication inhibition. In this study, a porcine scFv-phage display library was constructed and used as a tool for production of NSP1β-specific porcine scFvs (pscFvs). The pscFvs to NSP1β were linked to a cell-penetrating peptide to form cell-penetrating pscFvs (transbodies). The transbodies could internalize and inhibit PRRSV replication in the infected cells. Computerized-simulation indicated that the effective pscFvs used several residues in multiple complementarity determining regions (CDRs) to interact with many residues in the CLPro and C-terminal motifs, which might explain the mechanism of the pscFvs-mediated virus replication inhibition. Although experiments are needed to verify the anti-viral mechanism of the transbodies, current data provide evidence for developing the transbodies further for treatment and prevention of PRRSV infection.</jats:p

Humanized-Single Domain Antibodies (VH/V&lt;sub&gt;H&lt;/sub&gt;H) that Bound Specifically to&lt;em&gt; Naja kaouthia&lt;/em&gt; Phospholipase A2 and Neutralized the Enzymatic Activity

&lt;em&gt;Naja kaouthia&lt;/em&gt; (monocled cobra) venom contains many isoforms of secreted phospholipase A2 (sPLA&lt;sub&gt;2&lt;/sub&gt;). The PLA&lt;sub&gt;2&lt;/sub&gt; exerts several pharmacologic and toxic effects in the snake bitten subject, dependent or independent on the enzymatic activity. &lt;em&gt;N. kaouthia&lt;/em&gt; venom appeared in two protein profiles, P3 and P5, after fractionating the venom by ion exchange column chromatography. In this study, phage clones displaying humanized-camel single domain antibodies (VH/V&lt;sub&gt;H&lt;/sub&gt;H) that bound specifically to the P3 and P5 were selected from a humanized-camel VH/V&lt;sub&gt;H&lt;/sub&gt;H phage display library. Two phagemid transfected &lt;em&gt;E. coli&lt;/em&gt; clones (P3-1 and P3-3) produced humanized-V&lt;sub&gt;H&lt;/sub&gt;H, while another clone (P3-7) produced humanized-VH. At the optimal venom:antibody ratio, the VH/V&lt;sub&gt;H&lt;/sub&gt;H purified from the &lt;em&gt;E. coli&lt;/em&gt; homogenates neutralized PLA&lt;sub&gt;2&lt;/sub&gt; enzyme activity comparable to the horse immune serum against the &lt;em&gt;N. kaouthia&lt;/em&gt; holo-venom. Homology modeling and molecular docking revealed that the VH/V&lt;sub&gt;H&lt;/sub&gt;H covered the areas around the PLA&lt;sub&gt;2&lt;/sub&gt; catalytic groove and inserted their Complementarity Determining Regions (CDRs) into the enzymatic cleft. It is envisaged that the VH/V&lt;sub&gt;H&lt;/sub&gt;H would ameliorate/abrogate the principal toxicity of the venom PLA&lt;sub&gt;2&lt;/sub&gt; (membrane phospholipid catabolism leading to cellular and subcellular membrane damage which consequently causes hemolysis, hemorrhage, and dermo-/myo-necrosis), if they were used for passive immunotherapy of the cobra bitten victim. The speculation needs further investigations

Human Transbodies to HCV NS3/4A Protease Inhibit Viral Replication and Restore Host Innate Immunity

A safe and effective direct acting anti-hepatitis C virus (HCV) agent is still needed. In this study, human single-chain variable fragments of antibody (scFvs) that bound to HCV NS3/4A protein were produced by phage display technology. The engineered scFvs were linked to nonaarginines (R9) for making them cell penetrable. HCV RNA transfected-Huh7 cells treated with the transbodies produced from four different transformed E. coli clones had reduced HCV RNA inside the cells and in the cell spent media, as well as fewer HCV foci in the cell monolayer compared to the transfected cells in culture medium alone. The transbodies treated-transfected cells also had up-expression of the genes coding for the host innate immune response including TRIF, TRAF3, IRF3, IL-28B, and IFN-beta. Computerized homology modeling and intermolecular docking predicted that the effective transbodies interacted with several critical residues of the NS3/4A protease including those that form catalytic triads, oxyanion loop, and S1 and S6 pockets, as well as a zinc binding site. Although insight into molecular mechanisms of the transbodies need further laboratory investigation, it can be deduced from the current data that the transbodies blocked the HCV NS3/4A protease activities leading to the HCV replication inhibition and restoration of the virally suppressed host innate immunity. The engineered antibodies should be tested further for treatment of HCV infection either alone, in combination with current therapeutics, or in a mixture with their cognates specific to other HCV proteins

Human super antibody to viral RNA-dependent RNA polymerase produced by a modified Sortase self-cleave-bacteria surface display system

Abstract Background RNA-dependent RNA polymerase (RdRp) is a good target of anti-RNA virus agents; not only it is pivotal for the RNA virus replication cycle and highly conserved among RNA viruses across different families, but also lacks human homolog. Recently, human single-chain antibody (HuscFv) that bound to thumb domain of hepatitis C virus (HCV) RNA-dependent RNA polymerase (functionalized NS5B protein) was produced and engineered into cell-penetrating antibody (super antibody) in the form of cell-penetrating peptide (penetratin, PEN)-linked HuscFv (PEN-HuscFv34). The super antibody was produced and purified from inclusion body (IB) of a pen-huscfv34-vector-transformed Escherichia coli. The super antibody inhibited replication of alpha- and beta- coronaviruses, flaviviruses, and picornaviruses that were tested (broadly effective); thus, it has high potential for developing further towards a pan-anti-RNA virus agent. However, production, purification, and refolding of the super antibody molecules from the bacterial IB are laborious and hurdles to large-scale production. Therefore, in this study, Sortase-self-cleave method and bacteria surface display system were combined and modified for the super antibody production. Methods and results BL21 (DE3) ΔA E. coli, a strain lacking predominant outer membrane protein (OmpA) and ion and OmpT proteases, that displayed a membrane-anchored fusion protein, i.e., chimeric lipoprotein (Lpp′)-OmpA′, SUMO, Sortase protease, Sortase cleavage site (LPET↓G) and PEN-HuscFv34-6× His was generated. The soluble PEN-HuscFv34-6× His with glycine at the N-terminus could be released from the E. coli surface, simply by incubating the bacterial cells in a Sortase-cleavage buffer. After centrifugation, the G-PEN-HuscFv34-6× His could be purified from the supernatant. The purified G-PEN-HuscFv34-6× retained original cell-penetrating ability (being super antibody) and the broadly effective anti-RNA virus activity of the original IB-derived-PEN-HuscFv34. Conclusion The functionalized super antibody to RNA virus RdRp was successfully produced by using combined Sortase self-cleave and bacterial surface display systems with modification. The display system is suitable for downstream processing in a large-scale production of the super antibody. It is applicable also for production of other recombinant proteins in soluble free-folding form

Humanized-VHH Transbodies that Inhibit HCV Protease and Replication

There is a need for safe and broadly effective anti-HCV agents that can cope with genetic multiplicity and mutations of the virus. In this study, humanized-camel VHHs to genotype 3a HCV serine protease were produced and were linked molecularly to a cell penetrating peptide, penetratin (PEN). Human hepatic (Huh7) cells transfected with the JFH-1 RNA of HCV genotype 2a and treated with the cell penetrable nanobodies (transbodies) had a marked reduction of the HCV RNA intracellularly and in their culture fluids, less HCV foci inside the cells and less amounts of HCV core antigen in culture supernatants compared with the infected cells cultured in the medium alone. The PEN-VHH-treated-transfected cells also had up-regulation of the genes coding for the host innate immune response (TRIF, TRAF3, IRF3, IL-28B and IFN-β), indicating that the cell penetrable nanobodies rescued the host innate immune response from the HCV mediated-suppression. Computerized intermolecular docking revealed that the VHHs bound to residues of the protease catalytic triad, oxyanion loop and/or the NS3 N-terminal portion important for non-covalent binding of the NS4A protease cofactor protein. The so-produced transbodies have high potential for testing further as a candidate for safe, broadly effective and virus mutation tolerable anti-HCV agents