Identification of Conserved Linear Epitopes on Viral Protein 2 of Foot-and-Mouth Disease Virus Serotype O by Monoclonal Antibodies 6F4.D11.B6 and 8D6.B9.C3.

Foot-and-mouth disease (FMD) is a highly infectious disease of cloven-hoofed animals with a significant economic impact. Early diagnosis and effective prevention and control could reduce the spread of the disease which could possibly minimize economic losses. Epitope characterization based on monoclonal antibodies provide essential information for developing diagnostic assays and vaccine designs. In this study, monoclonal antibodies raised against FMD virus (FMDV) were produced. Sixty-six monoclonal antibodies demonstrated strong reactivity and specificity to FMDV. The purified monoclonal antibodies were further used for bio-panning to select phage expressing specific epitopes from phage-displayed 12 mer-peptide library. The phage peptide sequences were analyzed using multiple sequence alignment and evaluated by peptide ELISA. Two hybridoma clones secreted monoclonal antibodies recognizing linear epitopes on VP2 of FMDV serotype O. The non-neutralizing monoclonal antibody 6F4.D11.B6 recognized the residues 67-78 on antigenic site 2 resinding in VP2, while the neutralizing monoclonal antibody 8D6.B9.C3 recognized a novel linear epitope encompassing residues 115-126 on VP2. This information and the FMDV-specific monoclonal antibodies provide valuable sources for further study and application in diagnosis, therapeutics and vaccine designs to strengthen the disease prevention and control measures.

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

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.

Targeting Emerging RNA Viruses by Engineered Human Superantibody to Hepatitis C Virus RNA-Dependent RNA Polymerase.

RNA-dependent RNA polymerase (RdRp) is a unique and highly conserved enzyme across all members of the RNA virus superfamilies. Besides, humans do not have a homolog of this protein. Therefore, the RdRp is an attractive target for a broadly effective therapeutic agent against RNA viruses. In this study, a formerly generated cell-penetrating human single-chain antibody variable fragment (superantibody) to a conformational epitope of hepatitis C virus (HCV) RdRp, which inhibited the polymerase activity leading to the HCV replication inhibition and the host innate immunity restoration, was tested against emerging/reemerging RNA viruses. The superantibody could inhibit the replication of the other members of the Flaviviridae (DENV serotypes 1-4, ZIKV, and JEV), Picornaviridae (genus Enterovirus: EV71, CVA16), and Coronaviridae (genus Alphacoronavirus: PEDV, and genus Betacoronavirus: SARS-CoV-2 (Wuhan wild-type and the variants of concern), in a dose-dependent manner, as demonstrated by the reduction of intracellular viral RNAs and numbers of the released infectious particles. Computerized simulation indicated that the superantibody formed contact interfaces with many residues at the back of the thumb domain (thumb II site, T2) of DENV, ZIKV, JEV, EV71, and CVA16 and fingers and thumb domains of the HCV and coronaviruses (PEDV and SARS-CoV-2). The superantibody binding may cause allosteric change in the spatial conformation of the enzyme and disrupt the catalytic activity, leading to replication inhibition. Although the speculated molecular mechanism of the superantibody needs experimental support, existing data indicate that the superantibody has high potential as a non-chemical broadly effective anti-positive sense-RNA virus agent.

Animation as Supplementary Learning Material About Carcinogenic Liver Fluke in Classes for Primary Schoolchildren.

Carcinogenic liver fluke is still an issue of great concern in some countries of Southeast Asia, particularly in Thailand, Cambodia, the Lao People's Democratic Republic, and Vietnam. The infection, caused by Opisthorchis viverrini, is associated to cholangiocarcinoma and is endemic among human populations for whom raw fish is frequently consumed. Prevention and health education are required. Therefore, this study aimed to evaluate the effectiveness of educational intervention to improve knowledge among primary schoolchildren based on animation-assisted education. In this study, 80 participants (40 participants in the experimental group and 40 participants in the comparison group) were selected in 2018. The effectiveness of an interactive animation program in improving the knowledge of students studying liver fluke was determined based on scores on tests given before and immediately after completion of a 4.29-min animated program on the liver fluke life cycle, risk factors, disease, diagnosis, treatment, prevention, and control. Data were analyzed using SPSS-22 via paired t tests and independent samples t tests at a significance level of 0.05. A marked and significant improvement was observed in the immediate posttest compared with the pretest scores. More importantly, the students who had used the animated program achieved a significantly higher score on the final test than the comparison group. The results offered in the first report show that the use of the animated program facilitated education about liver fluke. It is strongly believed that animations are good supplementary learning materials for students, particularly for learning about serious concepts.

Publisher Correction: Human single chain-transbodies that bound to domain-I of non-structural protein 5A (NS5A) of hepatitis C virus.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Human single chain-transbodies that bound to domain-I of non-structural protein 5A (NS5A) of hepatitis C virus.

A safe and broadly effective direct acting anti-hepatitis C virus (HCV) agent that can withstand the viral mutation is needed. In this study, human single chain antibody variable fragments (HuscFvs) to conserved non-structural protein-5A (NS5A) of HCV were produced by phage display technology. Recombinant NS5A was used as bait for fishing-out the protein bound-phages from the HuscFv-phage display library. NS5A-bound HuscFvs produced by five phage transfected-E. coli clones were linked molecularly to nonaarginine (R9) for making them cell penetrable (become transbodies). The human monoclonal transbodies inhibited HCV replication in the HCVcc infected human hepatic cells and also rescued the cellular antiviral immune response from the viral suppression. Computerized simulation verified by immunoassays indicated that the transbodies used several residues in their multiple complementarity determining regions (CDRs) to form contact interface with many residues of the NS5A domain-I which is important for HCV replication complex formation and RNA binding as well as for interacting with several host proteins for viral immune evasion and regulation of cellular physiology. The human monoclonal transbodies have high potential for testing further as a new ramification of direct acting anti-HCV agent, either alone or in combination with their cognates that target other HCV proteins.

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-ß. 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.

Inhibition of HCV replication by humanized-single domain transbodies to NS4B.

NS4B of hepatitis C virus (HCV) initiates membrane web formation, binds RNA and other HCV proteins for viral replication complex (RC) formation, hydrolyses NTP, and inhibits innate anti-viral immunity. Thus, NS4B is an attractive target of a novel anti-HCV agent. In this study, humanized-nanobodies (VHs/VHHs) that bound to recombinant NS4B were produced by means of phage display technology. The nanobodies were linked molecularly to a cell penetrating peptide, penetratin (PEN), for making them cell penetrable (become transbodies). Human hepatic (Huh7) cells transfected with HCV JFH1-RNA that were treated with transbodies from four Escherichia coli clones (PEN-VHH7, PEN-VHH9, PEN-VH33, and PEN-VH43) had significant reduction of HCV RNA amounts in their culture fluids and intracellularly when compared to the transfected cells treated with control transbody and medium alone. The results were supported by the HCV foci assay. The transbody treated-transfected cells also had upregulation of the studied innate cytokine genes, IRF3, IFNß and IL-28b. The transbodies have high potential for testing further as a novel anti-HCV agent, either alone, adjunct of existing anti-HCV agents/remedies, or in combination with their cognates specific to other HCV enzymes/proteins.

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-ß), 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.

Cell penetrable human scFv specific to middle domain of matrix protein-1 protects mice from lethal influenza.

A new anti-influenza remedy that can tolerate the virus antigenic variation is needed. Influenza virus matrix protein-1 (M1) is highly conserved and pivotal for the virus replication cycle: virus uncoating, assembly and budding. An agent that blocks the M1 functions should be an effective anti-influenza agent. In this study, human scFv that bound to recombinant M1 middle domain (MD) and native M1 of A/H5N1 was produced. Phage mimotope search and computerized molecular docking revealed that the scFv bound to the MD conformational epitope formed by juxtaposed helices 7 and 9 of the M1. The scFv was linked molecularly to a cell penetrable peptide, penetratin (PEN). The PEN-scFv (transbody), when used to treat the cells pre-infected with the heterologous clade/subclade A/H5N1 reduced the viral mRNA intracellularly and in the cell culture fluids. The transbody mitigated symptom severity and lung histopathology of the H5N1 infected mice and caused reduction of virus antigen in the tissues as well as extricated the animals from the lethal challenge in a dose dependent manner. The transbody specific to the M1 MD, either alone or in combination with the cognate human scFvs specific to other influenza virus proteins, should be an effective, safe and mutation tolerable anti-influenza agent.

Interference of HCV replication by cell penetrable human monoclonal scFv specific to NS5B polymerase.

A new class of hepatitis C virus (HCV)-targeted therapeutics that is safe, broadly effective and can cope with virus mutations is needed. The HCV's NS5B is highly conserved and different from human protein, and thus it is an attractive target for anti-HCV therapeutics development. In this study, NS5B bound-phage clones selected from a human single chain variable antibody fragment (scFv) phage display library were used to transform appropriate E. coli bacteria. Two scFv inhibiting HCV polymerase activity were selected. The scFvs were linked to a cell penetrating peptide to make cell penetrable scFvs. The transbodies reduced the HCV RNA and infectious virus particles released into the culture medium and inside hepatic cells transfected with a heterologous HCV replicon. They also rescued the innate immune response of the transfected cells. Phage mimotope search and homology modeling/molecular docking revealed the NS5B subdomains and residues bound by the scFvs. The scFv mimotopes matched residues of the NS5B, which are important for nucleolin binding during HCV replication, as well as residues that interconnect the fingers and thumb domains for forming a polymerase active groove. Both scFvs docked on several residues at the thumb armadillo-like fold that could be the polymerase interactive sites of other viral/host proteins for the formation of the replication complex and replication initiation. In conclusion, human transbodies that inhibited HCV RdRp activity and HCV replication and restored the host innate immune response were produced. They are potentially future interferon-free anti-HCV candidates, particularly in combination with other cognates that are specific to NS5B epitopes and other HCV enzymes.

Ophiophagus hannah venom: proteome, components bound by Naja kaouthia antivenin and neutralization by N. kaouthia neurotoxin-specific human ScFv.

Venomous snakebites are an important health problem in tropical and subtropical countries. King cobra (Ophiophagus hannah) is the largest venomous snake found in South and Southeast Asia. In this study, the O. hannah venom proteome and the venom components cross-reactive to N. kaouthia monospecific antivenin were studied. O. hannah venom consisted of 14 different protein families, including three finger toxins, phospholipases, cysteine-rich secretory proteins, cobra venom factor, muscarinic toxin, L-amino acid oxidase, hypothetical proteins, low cysteine protein, phosphodiesterase, proteases, vespryn toxin, Kunitz, growth factor activators and others (coagulation factor, endonuclease, 5'-nucleotidase). N. kaouthia antivenin recognized several functionally different O. hannah venom proteins and mediated paratherapeutic efficacy by rescuing the O. hannah envenomed mice from lethality. An engineered human ScFv specific to N. kaouthia long neurotoxin (NkLN-HuScFv) cross-neutralized the O. hannah venom and extricated the O. hannah envenomed mice from death in a dose escalation manner. Homology modeling and molecular docking revealed that NkLN-HuScFv interacted with residues in loops 2 and 3 of the neurotoxins of both snake species, which are important for neuronal acetylcholine receptor binding. The data of this study are useful for snakebite treatment when and where the polyspecific antivenin is not available. Because the supply of horse-derived antivenin is limited and the preparation may cause some adverse effects in recipients, a cocktail of recombinant human ScFvs for various toxic venom components shared by different venomous snakes, exemplified by the in vitro produced NkLN-HuScFv in this study, should contribute to a possible future route for an improved alternative to the antivenins.

Humanized-VH/VHH that inhibit HCV replication by interfering with the virus helicase activity.

NS3 helicase is a pivotal enzyme involved in the early and late phases of hepatitis C virus (HCV) replication. The primary sequence and tertiary structure of this virus enzyme differ from human helicase to a certain extent; thus this virus protein has potential as a novel anti-HCV target. In this study, recombinant C-terminal NS3 protein of HCV genotype 3a with endowed helicase activity was produced and used as antigen by selecting VH/V(H)H display phage clones from an established humanized-camel single domain antibody library that bound specifically to HCV helicase. The VH/V(H)H derived from phage transfected Escherichia coli clones were linked molecularly to a cell penetrating peptide, i.e., penetratin (PEN). The cell penetrable VH/V(H)H (transbodies) could reduce the amounts of the HCV RNA released into the cell culture fluid and inside Huh7 cells infected with pJFH1 replicon with a greater effect on the former compared to the latter. Regions and residues of the helicase bound by the transbodies were determined by phage mimotope searching and multiple alignments as well as homology modeling and molecular docking. The epitope of one transbody (PEN-V(H)H9) encompassed residues 588RLKPTLHGPTPLLYRLGA605 of the domain 3 necessary for helicase activity while another transbody (PEN-VH59) interacted with the areas covering the phenylalanine loop and arginine clamp of the domain 2 which are important for the proper folding of the enzyme as well as nucleic acid substrate binding. Although the molecular mechanisms of the prototypic transbodies on NS3 helicase need further investigation, these transbodies have high potential as novel, safe and mutation tolerable anti-HCV agents.

Human monoclonal ScFv specific to NS1 protein inhibits replication of influenza viruses across types and subtypes.

Currently, there is a need of new anti-influenza agents that target influenza virus proteins other than ion channel M2 and neuraminidase. Non-structural protein-1 (NS1) is a highly conserved multifunctional protein which is indispensable for the virus replication cycle. In this study, fully human single chain antibody fragments (HuScFv) that bound specifically to recombinant and native NS1 were produced from three huscfv-phagemid transformed Escherichia coli clones (nos. 3, 10 and 11) selected from a human ScFv phage display library. Western blot analysis, mimotope searching/epitope identification, homology modeling/molecular docking and phage mimotope ELISA inhibition indicated that HuScFv of clone no. 3 reacted with NS1 R domain important for host innate immunity suppression; HuScFv of clone nos. 10 and 11 bound to E domain sites necessary for NS1 binding to the host eIF4GI and CPSF30, respectively. The HuScFv of all clones could enter the influenza virus infected cells and interfered with the NS1 activities leading to replication inhibition of viruses belonging to various heterologous A subtypes and type B by 2-64-fold as semi-quantified by hemagglutination assay. Influenza virus infected cells treated with representative HuScFv (clone 10) had up-expression of IRF3 and IFN-ß genes by 14.75 and 4.95-fold, respectively, in comparison with the controls, indicating that the antibodies could restore the host innate immune response. The fully human single chain antibodies have high potential for developing further as a safe (adjunctive) therapeutic agent for mitigating, if not abrogating, severe symptoms of influenza.

Human monoclonal ScFv that bind to different functional domains of M2 and inhibit H5N1 influenza virus replication.

BACKGROUND: Novel effective anti-influenza agent that tolerates influenza virus antigenic variation is needed. Highly conserved influenza virus M2 protein has multiple pivotal functions including ion channel activity for vRNP uncoating, anti-autophagy and virus assembly, morphogenesis and release. Thus, M2 is an attractive target of anti-influenza agents including small molecular drugs and specific antibodies. METHODS: Fully human monoclonal single chain antibodies (HuScFv) specific to recombinant and native M2 proteins of A/H5N1 virus were produced from huscfv-phagemid transformed E. coli clones selected from a HuScFv phage display library using recombinant M2 of clade 1 A/H5N1 as panning antigen. The HuScFv were tested for their ability to inhibit replication of A/H5N1 of both homologous and heterologous clades. M2 domains bound by HuScFv of individual E. coli clones were identified by phage mimotope searching and computerized molecular docking. RESULTS: HuScFv derived from four huscfv-phagemid transformed E. coli clones (no. 2, 19, 23 and 27) showed different amino acid sequences particularly at the CDRs. Cells infected with A/H5N1 influenza viruses (both adamantane sensitive and resistant) that had been exposed to the HuScFv had reduced virus release and intracellular virus. Phage peptide mimotope search and multiple alignments revealed that conformational epitopes of HuScFv2 located at the residues important for ion channel activity, anti-autophagy and M1 binding; epitopic residues of HuScFv19 located at the M2 amphipathic helix and cytoplasmic tail important for anti-autophagy, virus assembly, morphogenesis and release; epitope of HuScFv23 involved residues important for the M2 activities similar to HuScFv2 and also amphipathic helix residues for viral budding and release while HuScFv27 epitope spanned ectodomain, ion channel and anti-autophagy residues. Results of computerized homology modelling and molecular docking conformed to the epitope identification by phages. CONCLUSIONS: HuScFv that bound to highly conserved epitopes across influenza A subtypes and human pathogenic H5N1clades located on different functional domains of M2 were produced. The HuScFv reduced viral release and intracellular virus of infected cells. While the molecular mechanisms of the HuScFv await experimental validation, the small human antibody fragments have high potential for developing further as a safe, novel and mutation tolerable anti-influenza agent especially against drug resistant variants.

Cell penetrable humanized-VH/V(H)H that inhibit RNA dependent RNA polymerase (NS5B) of HCV.

NS5B is pivotal RNA dependent RNA polymerase (RdRp) of HCV and NS5B function interfering halts the virus infective cycle. This work aimed to produce cell penetrable humanized single domain antibodies (SdAb; VH/V(H)H) that interfere with the RdRp activity. Recombinant NS5BΔ55 of genotype 3a HCV with de novo RNA synthetic activity was produced and used in phage biopanning for selecting phage clones that displayed NS5BΔ55 bound VH/V(H)H from a humanized-camel VH/V(H)H display library. VH/V(H)H from E. coli transfected with four selected phage clones inhibited RdRp activity when tested by ELISA inhibition using 3'di-cytidylate 25 nucleotide directed in vitro RNA synthesis. Deduced amino acid sequences of two clones showed V(H)H hallmark and were designated V(H)H6 and V(H)H24; other clones were conventional VH, designated VH9 and VH13. All VH/V(H)H were linked molecularly to a cell penetrating peptide, penetratin. The cell penetrable VH9, VH13, V(H)H6 and V(H)H24 added to culture of Huh7 cells transfected with JHF-1 RNA of genotype 2a HCV reduced the amounts of RNA intracellularly and in culture medium implying that they inhibited the virus replication. VH/V(H)H mimotopes matched with residues scattered on the polymerase fingers, palm and thumb which were likely juxtaposed to form conformational epitopes. Molecular docking revealed that the antibodies covered the RdRp catalytic groove. The transbodies await further studies for in vivo role in inhibiting HCV replication.

Humanized-single domain antibodies (VH/VHH) that bound specifically to Naja kaouthia phospholipase A2 and neutralized the enzymatic activity.

Naja kaouthia (monocled cobra) venom contains many isoforms of secreted phospholipase A2 (sPLA(2)). The PLA(2) exerts several pharmacologic and toxic effects in the snake bitten subject, dependent or independent on the enzymatic activity. N. kaouthia 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(H)H) that bound specifically to the P3 and P5 were selected from a humanized-camel VH/V(H)H phage display library. Two phagemid transfected E. coli clones (P3-1 and P3-3) produced humanized-V(H)H, while another clone (P3-7) produced humanized-VH. At the optimal venom:antibody ratio, the VH/V(H)H purified from the E. coli homogenates neutralized PLA(2) enzyme activity comparable to the horse immune serum against the N. kaouthia holo-venom. Homology modeling and molecular docking revealed that the VH/V(H)H covered the areas around the PLA(2) catalytic groove and inserted their Complementarity Determining Regions (CDRs) into the enzymatic cleft. It is envisaged that the VH/V(H)H would ameliorate/abrogate the principal toxicity of the venom PLA(2) (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.

Heterosubtypic immunity to influenza mediated by liposome adjuvanted H5N1 recombinant protein vaccines.

A non-egg, non-culture based influenza vaccine that intervenes large influenza outbreaks and protects against heterosubtypic infections is needed. Candidates of such vaccine are likely to be conserved influenza virus proteins or their coding DNA. The vaccine must be conveniently produced at reasonable cost, safe, highly immunogenic and should be able to recall rapidly the immunological memory upon the antigenic re-exposure. In this study vaccines made of full length recombinant NP and M2 of the H5N1 influenza A virus were entrapped either alone or together into liposome (L) made of phosphatidylcholine and cholesterol. The vaccines (L-NP, L-M2 or L-NP+M2) and mocks (L or PBS) were safe without causing any adverse reaction in the intramuscularly injected mice. They were readily immunogenic at a single dose and a recalled response could be detected within one day post booster. Cytokine and antibody data indicated that the vaccines induced a Th1 bias immune response. NP containing vaccines stimulated a marked increase of cytotoxic lymphocytes, i.e., CD8(+), intracellular IFNγ(+) cells, while M2 containing vaccines elicited good antibody response which neutralized infectivity of heterologous influenza viruses. Although the three vaccines elicited different immunological defense factors; nevertheless, they similarly and readily abrogated lung histopathology mediated by viruses belonging to different H5N1 clade/subclade and heterosubtypes including swine H1N1 and human H1N1/2009 viruses. They protected the vaccinated mice against lethal challenges with mouse adapted avian H5N1 virus. The liposome adjuvanted vaccines which demonstrated high protective efficacy in mice warrant testing further in a non-rodent model as well as in humans.

Human single-chain antibodies that neutralize homologous and heterologous strains and clades of influenza A virus subtype H5N1.

BACKGROUND: Human antibodies that interfere with the biological activity of haemagglutinins (HAs) of influenza viruses have high potential as an antiviral agent. METHODS: Human single-chain antibody fragments (HuScFv) to recombinant and native HAs of the influenza virus H5N1 subtype were produced using a human antibody phage display library with the intention to increase the therapeutic arsenal against this highly pathogenic virus. RESULTS: The HuScFv inhibited HA activity and neutralized infectivity of both homologous and heterologous strains and clades of the H5N1 subtype in Madin-Darby canine kidney cell cultures. Intraperitoneally injected HuScFv also mediated immunotherapeutic protection in mice that were intranasally challenged with highly pathogenic H5N1 viruses belonging to different strains and clades. CONCLUSIONS: Our data indicate that it might be worth pursuing these HuScFv further for future consideration as candidates for influenza intervention and treatment.

Human single chain monoclonal antibody that recognizes matrix protein of heterologous influenza A virus subtypes.

Matrix protein (M1) is predominant and has pivotal role in the influenza A virus replication and assembly. It is therefore an attractive target for antiviral drugs, siRNA studies, and therapeutic antibodies. Nevertheless, therapeutic antibody that interferes with the M1 multiplex function has never been developed. In this study, human single monoclonal antibody fragments (HuScFvs) to M1 were generated. Full length recombinant M1 (rM1) was produced from cDNA prepared from genome of highly pathogenic avian influenza virus, A/H5N1. The rM1 was used as an antigen in phage bio-panning to select phage clones displaying HuScFv from a human antibody phage display library. Several phage clones displaying HuScFv bound to the rM1 and harboring the respective huscfv gene inserts were isolated. RFLP experiments revealed multiple DNA banding patterns which indicated epitope/affinity diversity of the HuScFv. The HuScFv were tested for their binding to native M1 of homologous and heterologous influenza A viruses using ELISA as well as incorporating immunostaining and immunofluorescence studies with infected MDCK cells. One such protein produced from a selected phage clone blocked binding of M1 to viral RNA. The HuScFv in their in vivo functional format, e.g. cell-penetrating molecules, should be developed and tested as a broad spectrum anti-A/influenza.