Mimotopes selected with neutralizing antibodies against multiple subtypes of influenza A

<p>Abstract</p> <p>Background</p> <p>The mimotopes of viruses are considered as the good targets for vaccine design. We prepared mimotopes against multiple subtypes of influenza A and evaluate their immune responses in flu virus challenged Balb/c mice.</p> <p>Methods</p> <p>The mimotopes of influenza A including pandemic H1N1, H3N2, H2N2 and H1N1 swine-origin influenza virus were screened by peptide phage display libraries, respectively. These mimotopes were engineered in one protein as multi- epitopes in Escherichia coli (E. coli) and purified. Balb/c mice were immunized using the multi-mimotopes protein and specific antibody responses were analyzed using hemagglutination inhibition (HI) assay and enzyme-linked immunosorbent assay (ELISA). The lung inflammation level was evaluated by hematoxylin and eosin (HE).</p> <p>Results</p> <p>Linear heptopeptide and dodecapeptide mimotopes were obtained for these influenza virus. The recombinant multi-mimotopes protein was a 73 kDa fusion protein. Comparing immunized infected groups with unimmunized infected subsets, significant differences were observed in the body weight loss and survival rate. The antiserum contained higher HI Ab titer against H1N1 virus and the lung inflammation level were significantly decreased in immunized infected groups.</p> <p>Conclusions</p> <p>Phage-displayed mimotopes against multiple subtypes of influenza A were accessible to the mouse immune system and triggered a humoral response to above virus.</p

Virus-Like Particles of SARS-Like Coronavirus Formed by Membrane Proteins from Different Origins Demonstrate Stimulating Activity in Human Dendritic Cells

The pathogenesis of SARS coronavirus (CoV) remains poorly understood. In the current study, two recombinant baculovirus were generated to express the spike (S) protein of SARS-like coronavirus (SL-CoV) isolated from bats (vAcBS) and the envelope (E) and membrane (M) proteins of SARS-CoV, respectively. Co-infection of insect cells with these two recombinant baculoviruses led to self-assembly of virus-like particles (BVLPs) as demonstrated by electron microscopy. Incorporation of S protein of vAcBS (BS) into VLPs was confirmed by western blot and immunogold labeling. Such BVLPs up-regulated the level of CD40, CD80, CD86, CD83, and enhanced the secretion of IL-6, IL-10 and TNF-α in immature dendritic cells (DCs). Immune responses were compared in immature DCs inoculated with BVLPs or with VLPs formed by S, E and M proteins of human SARS-CoV. BVLPs showed a stronger ability to stimulate DCs in terms of cytokine induction as evidenced by 2 to 6 fold higher production of IL-6 and TNF-α. Further study indicated that IFN-γ+ and IL-4+ populations in CD4+ T cells increased upon co-cultivation with DCs pre-exposed with BVLPs or SARS-CoV VLPs. The observed difference in DC-stimulating activity between BVLPs and SARS CoV VLPs was very likely due to the S protein. In agreement, SL-CoV S DNA vaccine evoked a more vigorous antibody response and a stronger T cell response than SARS-CoV S DNA in mice. Our data have demonstrated for the first time that SL-CoV VLPs formed by membrane proteins of different origins, one from SL-CoV isolated from bats (BS) and the other two from human SARS-CoV (E and M), activated immature DCs and enhanced the expression of co-stimulatory molecules and the secretion of cytokines. Finding in this study may provide important information for vaccine development as well as for understanding the pathogenesis of SARS-like CoV

Induction of significant neutralizing antibodies against SARS-CoV-2 by a highly attenuated pangolin coronavirus variant with a 104nt deletion at the 3'-UTR

ABSTRACTSARS-CoV-2 related coronaviruses (SARS-CoV-2r) from Guangdong and Guangxi pangolins have been implicated in the emergence of SARS-CoV-2 and future pandemics. We previously reported the culture of a SARS-CoV-2r GX_P2V from Guangxi pangolins. Here we report the GX_P2V isolate rapidly adapted to Vero cells by acquiring two genomic mutations: an alanine to valine substitution in the nucleoprotein and a 104-nucleotide deletion in the hypervariable region (HVR) of the 3′-terminus untranslated region (3′-UTR). We further report the characterization of the GX_P2V variant (renamed GX_P2V(short_3UTR)) in in vitro and in vivo infection models. In cultured Vero, BGM and Calu-3 cells, GX_P2V(short_3UTR) had similar robust replication kinetics, and consistently produced minimum cell damage. GX_P2V(short_3UTR) infected golden hamsters and BALB/c mice but was highly attenuated. Golden hamsters infected intranasally had a short duration of productive infection in pulmonary, not extrapulmonary, tissues. These productive infections induced neutralizing antibodies against pseudoviruses of GX_P2V and SARS-CoV-2. Collectively, our data show that the GX_P2V(short_3UTR) is highly attenuated in in vitro and in vivo infection models. Attenuation of the variant is likely partially due to the 104-nt deletion in the HVR in the 3′-UTR. This study furthers our understanding of pangolin coronaviruses pathogenesis and provides novel insights for the design of live attenuated vaccines against SARS-CoV-2

Cytokines secretions in DCs treated with BVLPs or with SARS CoV VLPs.

<p>DCs were incubated with SL-CoV BVLPs or SARS CoV VLPs at a concentration of 10 µg/ml. DCs treated with LPS (10 µg/ml) and PBS were used as positive and negative control, respectively. After 16 h incubation, supernatants were collected, and IL-6, IL10 and TNF-α were analyzed using ELISA. Data are expressed as the mean±SD of triplicate samples.</p

Confirmation of the incorporation of BS protein in BVLPs.

<p>(A) Budding of BVLPs from insect cells. Sf21 cells were co-infected with recombinant baculovirus vAcBS and vAcME at a moi of 5. At 72 h post-infection, cells were harvested and fixed with 2% glutaraldehyde and then with 1% osmium tetroxide. Thin-section samples were prepared and stained with 1% uranyl acetate followed by examination under a transmission electron microscope. Arrows indicates BVLPs. Bar = 250 nm. (B) Western blot analysis of BVLPs. 5 µg purified BVLPs were analyzed by western blot using rabbit-derived anti-SARS-CoV antibody. An equivalent amount of purified human SARS CoV VLPs was run in parallel. Preimmune rabbit antiserum was used as negative control. Left part, band corresponds to S protein. Lane 1, negative control; lane 2, human SARS CoV VLPs; lane 3, BVLPs. Right part, band corresponds to E or M protein. Lane 1, human SARS CoV VLPs; lane 2, negative control; lane 3, BVLPs. The protein marker was Fermentas #SM0671. (C) Detection of BVLPs by immunogold labeling. The collodion-coated EM grids were loaded with purified BVLPs (left), SARS CoV VLPs (middle) or VLPs without BS or S protein (right) for 5 min. After removal of the excess sample solution, grids were incubated with antibody specific against BS2 protein for 1 h and then incubated with 15 nm gold conjugated anti-rabbit IgG.. The samples were stained with 2% PTA for 1 min, drained and examined under the EM. Arrow indicates the gold particles and triangle indicates the VLPs. Bar = 100 nm.</p

Dissemination of KPC-2-Encoding IncX6 Plasmids Among Multiple Enterobacteriaceae Species in a Single Chinese Hospital

Forty-five KPC-producing Enterobacteriaceae strains were isolated from multiple departments in a Chinese public hospital from 2014 to 2015. Genome sequencing of four representative strains, namely Proteus mirabilis GN2, Serratia marcescens GN26, Morganella morganii GN28, and Klebsiella aerogenes E20, indicated the presence of blaKPC-2-carrying IncX6 plasmids pGN2-KPC, pGN26-KPC, pGN28-KPC, and pE20-KPC in the four strains, respectively. These plasmids were genetically closely related to one another and to the only previously sequenced IncX6 plasmid, pKPC3_SZ. Each of the plasmids carried a single accessory module containing the blaKPC-2/3-carrying ΔTn6296 derivatives. The ΔTn6292 element from pGN26-KPC also contained qnrS, which was absent from all other plasmids. Overall, pKPC3_SZ-like blaKPC-carrying IncX6 plasmids were detected by PCR in 44.4% of the KPC-producing isolates, which included K. aerogenes, P. mirabilis, S. marcescens, M. morganii, Escherichia coli, and Klebsiella pneumoniae, and were obtained from six different departments of the hospital. Data presented herein provided insights into the genomic diversity and evolution of IncX6 plasmids, as well as the dissemination and epidemiology of blaKPC-carrying IncX6 plasmids among Enterobacteriaceae in a hospital setting

Detection of SARS-CoV S-specific IgG and the subclasses in vaccinated mice.

<p>Mouse sera (8 per group) were collected 10 days after the final immunization and assayed for IgG1 and IgG2a against the S2 protein of SARS-CoV. ELISA was used to detect the level of S-specific IgG antibodies. Recombinant S2 protein expressed in <i>E. Coli</i> was purified and used as the detection antigen. Data are presented as means±SD.</p