Screening And Functional Study Of Nanobodies Against Newcastle Disease Virus Haemagglutinin-Neuraminidase Protein

Newcastle disease(ND), which is caused by the Newcastle disease virus(NDV), is one of the most highly contagious avian diseases that affect chicken and other birds. Since its first report in 1926, ND had four times prevalence worldwide and caused great economic losses to the poultry industry. Now, ND remains a great threat to chickens and other wild birds and is listed as a notifiable animal disease by Office International Des Epizooties(OIE). In the past decades, ND epidemics were effectively controlled because of widespread vaccination policy adopted by many countries around the world. Whereas, ND outbreaks still occur, and virulent NDV is sporadically isolated from vaccinated chickens and wild birds. A considerable number of studies indicated that current vaccines and therapeutic antibody-like biological agents could not completely stop the transmission of virulent NDVs. Therefore, the development of novel method for ND control is necessary.In the late 1990 s, Hamers reported the discovery of heavy-chain antibody in camels and sharks for the first time. Variable domains of camelid heavy-chain antibodies(VHH), which derived from camelid heavy-chain antibody, are the smallest natural occurring functional antibody fragments that maintain the antigen-binding capacity. Due to their single domain nature and small size(only 15 kDa), with a height of 4 nm and diameter of 2 nm, VHH are also called nanobodies(Nbs). The analysis of it's structure revealed that all VHH share a few crucial hydrophilic amino acids substitutions at position 37, 44, 45 and 47 in FR2, and there are also additional Cys residues in CDR1 and CDR3. Furthermore, compared to conventional VH domains, VHH often display a longer CDR3 loop. The above structure characteristics make VHH relatively stable, share high affinity, not easy to aggregation and could bind epitopes inaccessible to conventional antibodies. Therefore, VHH are ideal candidates for a considerable number of virus disease therapeutic and diagnostic applications. However, an anti-NDV VHH has not been reported to date.To acquire anti-NDV VHH in the present study, we constructed an NDV immunized VHH yeast two-hybrid library and the library quality was evaluated at first. Subsequently, the truncate HN protein was used as target to construct bait plasmid pGBKT7-HN. Then VHH yeast two-hybrid library was screened using Y2 H Gold bait strain that transformed with pGBKT7-HN. The selected positive clones were expressed and purified. The reactivitybetween NDV and VHH were further identified. At last, a dimerized VHH was constructed to improve the activity of VHH. The main results are as follows:(1) A six-month-old female C. Bactrianus was immunized with a combination of inactivated NDV(La Sota) and subtype H9 avian influenza(Strain F) vaccine five times at two-weeks intervals. The administration dose was based on the weight ratio between chicken and C. Bactrianus. After vaccination, the humoral immune response was monitored by HI assay. The animal with a strong response was bled for peripheral blood lymphocytes(PBL)isolation. Then total RNA was extracted from collected PBL, and the first-strand cDNA was synthesized. Two rounds PCR were successfully performed to amplify VHH fragments. The VHH fragments with homologous arms and linearized pGADT7-Rec plasmid were cotransformed into Y187 yeast competent cells to construct the VHH yeast two-hybrid library.Four parameters were used to evaluated the quality of the yeast two-hybrid library, results of the calculations were as follows: the library capacity was 1.25×107, the library titer was3.45×108 cfu/mL, the library insertion rate was approximately 96%. Ten PCR positive clones were chosen randomly for sequencing, results showed that these clones were all unique sequences, indicating diversity of the library.(2) Using NDV HN protein as target, the truncate HN gene without transmembrane region was successfully amplified. Then the amplified VHH was cloned into pGBKT7 vector and designed as pGBKT7-HN. Y2 H Gold strain transformed with pGBKT7-HN was test against autoactivator activity and toxicity before screening, result showed that the bait strain had no autoactivator activity and toxicity. Subsequently, the VHH yeast two-hybrid library was screened, and the prey plasmids were recovered from positive clones. The recovered prey plasmids and bait plasmid were co-transformed into Y2 H Gold yeast strain to confirm the genuine interaction in yeast. The recovered genuine positive clones were sequenced and aligned. Finally, seven positive clones were acquired.(3) The seven screened VHH fragments were amplified and cloned into prokaryotic expression vector pHSIE for fusion with 6×His-SUMO tag at the N-terminus and designed as pHSIE-VHH. Then, the recombinant pHSIE-VHH were transformed into E.coli Rosetta for expression. And the expression conditions were optimized, results showed that the optimized expression condition were induction for 6 h by adding 0.4 mmol/L IPTG. After large scale induction for expression, the recombinant VHH were purified according to the Talon Metal Affinity Resin user manual(Clontech). At the same time, the purification condition was optimized. Finally, we successfully acquired pure recombinant VHH antibodies with few irrelevant protein. To further detect the reactivity between purified VHH and NDV virons or HN protein, HI assay, indirect ELISA, dot blot, pull down, immunocytochemistry andneutralizing assay were performed. HI assay results showed that the seven VHH antibodies could inhibit the haemagglutination activity of different NDV strains at different levels, but the HI titers were significantly lower than that of positive control(P < 0.05). Indirect ELISA results suggested that all purified VHH could interact with NDV viron and the reactivity of VHH3, VHH4, VHH5, VHH6 and VHH7 were higher than positive control. Dot blot results further demonstrated that the seven VHHs were able to recognize naive NDV viron.Immunocytochemistry results indicated the feasibility of detection intracellular NDV antigen by using selected VHH. In addition, five VHH antibodies had partial neutralizing activity and could inhibit the replication of NDV on DF-1 cell at the early stage of infection.(4) To improve the reactivity and neutralizing activity of VHH, a screened VHH was tandem repeated twice by a flexible linker to construct a dimerized VHH through overlap PCR, the constructed expression vector was designed as pHSIE-Dim VHH. The pHSIE-Dim VHH was transformed into Rosetta competent cells for expression and the expression condition was optimized. Results showed that the best expression condition were induction for 12 h by adding 0.4 mmol/L IPTG. The expressed recombinant dimerized VHH was purified according to Talon Metal affinity Resin user manual(Clontech) and the purification condition was optimized. We finally obtained relatively pure dimerized VHH. Indirect ELISA results showed that the reactivity of dimerized VHH was higher than that of monovalent VHH,but the neutralizing activity had no increase.(5) Intrabody was an ideal candidate for anti-viral and metabolism regulation research.As a form of intrabody, great attention was paid to nanobody. To study the anti-viral activity of our selected VHH, we constructed VHH lentivirus expression vector. After transfection with package helper plasmids, a lentivirus, named Lenti-VHH, was rescued. Then a DF-1 cell line that could stable express VHH was established by lentivirus transduction after puromycine selection. Results showed that stable expression VHH could inhibit the proliferation.