Influenza A virus nucleoprotein derived from Escherichia coli or recombinant vaccinia (Tiantan) virus elicits robust cross-protection in mice.

BACKGROUND: Immunity to conserved viral antigens is an attractive approach to develop a universal vaccine against epidemic and pandemic influenza. A nucleoprotein (NP)-based vaccine has been explored and preliminary studies have shown promise. However, no study has explored the immunity and cross-protective efficacy of recombinant NP derived from Escherichia coli compared with recombinant vaccinia virus (Tiantan). METHODS: Recombinant NP protein (rNP) from influenza virus A/Jingke/30/95(H3N2) was obtained from E. coli and recombinant vaccinia virus (Tiantan) RVJ1175NP. Purified rNP without adjuvant and RVJ1175NP were used to immunize BALB/c mice intramuscularly. Humoral immune responses were detected by ELISA, while cell-mediated immune responses were measured by ex vivo IFN-γ ELISPOT and in vivo cytotoxicity assays. The cross-protective efficacy was assessed by a challenge with a heterosubtype of influenza virus A/PR/8/34(H1N1). RESULTS: Our results demonstrate that a high dose (90 µg) of rNP induced NP-specific antibodies and T cell responses that were comparable with those of RVJ1175NP in mice. Importantly, the survival ratio (36, 73, and 78%) of the vaccinated mice after the influenza virus A/PR/8/34(H1N1) challenge was rNP vaccine dose-dependent (10, 30, and 90 µg, respectively), and no significant differences were observed between the rNP- and RVJ1175NP-immunized (91%) mice. CONCLUSIONS: Influenza A virus NP derived from E. coli or recombinant vaccinia (Tiantan) virus elicited cross-protection against influenza virus in mice, and the immune response and protective efficacy of rNP were comparable to RVJ1175NP. These data provide a basis for the use of prokaryotically expressed NP as a candidate universal influenza vaccine.

Three doses of an inactivation-based COVID-19 vaccine induces cross-neutralizing immunity against the SARS CoV-2 Omicron variant.

The immunity potency upon natural infection or vaccination is the main concern for the vaccine strategy of severe acute respiratory syndrome coronavirus 2 (SARS COV-2 variant), especially the recently reported Omicron variant (B.1.1.529). In this study, 200 recipients immunized with three doses of a COVID-19-inactivated vaccine were enrolled, whose serum samples were collected within 2 months after the third immunization. The neutralizing activity of sera against the pseudotyped Omicron variant, prototype, and Delta variant was determined. Our results demonstrated that the positive neutralization activity was 95.5% for the Omicron variant, 99.5% for the prototype, and 98.5% for the Delta variant. The geometric mean titers (GMT) for the Omicron variant was 49 and maintained sustained immune levels for 2 months, which decreased by 4.9-fold and 3.0-fold compared with the prototype (GMT, 239) and Delta variant (GMT, 148), respectively. In summary, our study demonstrated that three doses of a COVID-19-inactivated vaccine effectively yielded potent cross-neutralizing activity against the Omicron variant at 2 months after the third vaccination.

Mechanism of Yifei Decoction Combined with MitoQ on Inhibition of TGFß1/NOX4 and PDGF/ROCK Signal Pathway in Idiopathic Pulmonary Fibrosis.

BACKGROUND: Rho-related coiled helix forming protein kinase (Rho-ROCK) and another important fibrogenic factor-PDGF play a critical role in collagen deposition in rat lung tissue. Yifei decoction (YFT), a Chinese herbal decoction, has been used to treat idiopathic pulmonary fibrosis (IPF) in clinical practice and has produced positive outcomes; however, convincing evidence is currently lacking. The present study aimed to investigate the effects of YFT combined with MitoQ in rats with IPF and to explore the underlying mechanism. METHODS: Rat IPF model was established by endotracheal injection of 5 mg/kg BleomycinA5 into the specific pathogen-free SD rats. MitoQ (6.5 µmol/kg once daily), YFT (10 ml/kg once daily), and MitoQ + YFT (6.5 µmol/kg + 10 ml/kg once daily) were used to treat the rat model for 4 weeks, respectively. The normal rats without IPF were used as the controls. After 4 weeks of drug treatment, lung histopathology was assessed. Immunohistochemistry was used to detect the expression of fibronectin and collagen IV in lung tissue. The expression of IL-6, IL-1ß, TNF-α, GSH-Px, SOD, MDA, and hydroxyproline was determined by enzyme-linked immunosorbent assay. The expressions of TGFß1, NOX4, PDGFR-ß, and ROCK1 were determined using real-time quantitative PCR and Western blot. RESULTS: After 4 weeks of drug treatment, comparison of the MitoQ + YFT group with the IPF group showed that lung injury scores, W/D, lung tissue hydroxyproline, fibronectin, collagen IV content, and IL-6, IL-1ß, TNF-α, and MDA levels were significantly lower (P < 0.05), as well as the expression of TGFß1, NOX4, PDGFR-ß, and ROCK1, but the activity of GSH-Px and SOD was higher (P < 0.05). CONCLUSION: MitoQ combined with YFT can improve lung injury in rats with pulmonary fibrosis by reducing the secretion of proinflammatory cytokines and inhibiting TGFß1/NOX4 and PDGF/ROCK signaling pathways. It may provide a new method for the treatment of pulmonary fibrosis.

Radioresistance of the breast tumor is highly correlated to its level of cancer stem cell and its clinical implication for breast irradiation.

BACKGROUND AND PURPOSE: Growing evidence suggested the coexistence of cancer stem cells (CSCs) within solid tumors. We aimed to study radiosensitivity parameters for the CSCs and differentiated tumor cells (TCs) and the correlation of the fractions of CSCs to the overall tumor radioresistance. MATERIAL AND METHODS: Surviving fractions of breast cancer cell lines were analyzed using a dual-compartment Linear-quadratic model with independent fitting parameters: radiosensitive αTC, ßTC, αCSC, ßCSC, and fraction of CSCs f. The overall tumor radio-resistance, the biological effective doses and tumor control probability were estimated as a function of CSC fraction for different fractionation regimens. The pooled clinical outcome data were fitted to the single- and dual-compartment linear-quadric models. RESULTS: CSCs were more radioresistant characterized by smaller α compared to TCs: αTC=0.1±0.2, αCSC=0.04±0.07 for MCF-7 (f=0.1%), αTC=0.08±0.25, αCSC=0.04±0.18 for SUM159PT (f=2.46%). Higher f values were correlated with increasing radioresistance in cell lines. Analysis of clinical outcome data is in accordance of a dual-compartment CSC model prediction. Higher percentage of BCSCs resulted in more overall tumor radioresistance and less biological effectiveness. CONCLUSIONS: Percentage of CSCs strongly correlated to overall tumor radioresistance. This observation suggested potential individualized radiotherapy to account for heterogeneous population of CSCs and their distinct radiosensitivity for breast cancer.

Protective Efficacy of the Conserved NP, PB1, and M1 Proteins as Immunogens in DNA- and Vaccinia Virus-Based Universal Influenza A Virus Vaccines in Mice.

The conventional hemagglutinin (HA)- and neuraminidase (NA)-based influenza vaccines need to be updated most years and are ineffective if the glycoprotein HA of the vaccine strains is a mismatch with that of the epidemic strain. Universal vaccines targeting conserved viral components might provide cross-protection and thus complement and improve conventional vaccines. In this study, we generated DNA plasmids and recombinant vaccinia viruses expressing the conserved proteins nucleoprotein (NP), polymerase basic 1 (PB1), and matrix 1 (M1) from influenza virus strain A/Beijing/30/95 (H3N2). BALB/c mice were immunized intramuscularly with a single vaccine based on NP, PB1, or M1 alone or a combination vaccine based on all three antigens and were then challenged with lethal doses of the heterologous influenza virus strain A/PR/8/34 (H1N1). Vaccines based on NP, PB1, and M1 provided complete or partial protection against challenge with 1.7 50% lethal dose (LD50) of PR8 in mice. Of the three antigens, NP-based vaccines induced protection against 5 LD50 and 10 LD50 and thus exhibited the greatest protective effect. Universal influenza vaccines based on the combination of NP, PB1, and M1 induced a strong immune response and thus might be an alternative approach to addressing future influenza virus pandemics.

Maximal immune response and cross protection by influenza virus nucleoprotein derived from E. coli using an optimized formulation.

The highly conserved internal nucleoprotein (NP) is a promising antigen to develop a universal influenza A virus vaccine. In this study, mice were injected intramuscularly with Escherichia coli-derived NP protein alone or in combination with adjuvant alum (Al(OH)3), CpG or both. The results showed that the NP protein formulated with adjuvant was effective in inducing a protective immune response. Additionally, the adjuvant efficacy of Al(OH)3 was stronger than that of CpG. Optimal immune responses were observed in BALB/c mice immunized with a combination of NP protein plus Al(OH)3 and CpG. These mice also showed maximal resistance following challenge with influenza A virus PR8 strain. Most importantly, 10 µg NP formulated with Al(OH)3 and CpG induced higher protection than did 90 µg NP. These findings indicated that a combination of Al(OH)3 and CpG may be an efficient adjuvant in the NP formulation.

Recombinant DNA vaccine against neurite outgrowth inhibitors attenuates behavioral deficits and decreases Abeta in an Alzheimer's disease mouse model.

Alzheimer's disease (AD) is a chronic neurodegenerative disease that causes a progressive loss in learning and memory capabilities and eventually results in dementia. The non-renewable nature of neurons in the central nervous system leads to the basic pathological changes that are related to the various behavioral and psychological symptoms of AD. Oligodendrocyte- and myelin-related neurite outgrowth inhibitors (NOIs) tend to hinder the regeneration of neurons. We designed a recombinant DNA vaccine composed of multiple specific inhibitory domains of NOIs. Vaccination induced effective antibodies against the specific domains in the sera of mice treated with a DNA primed-vaccinia virus boost regimen. The vaccine attenuated neuronal degeneration in the mouse brain and protected the model mice from behavioral deficits. Vaccination also decreased the formation of soluble Aß oligomer and amyloid plaques in the co-transgenic mice brain. What's more, astrocytosis in brains of APP/PS1 co-transgenic mice was also relieved. The results suggested that immunotherapy with multiple specific domains of myelin- and oligodendrocyte-related NOIs may be a promising approach for Alzheimer's disease and other degenerative central nervous system diseases.

Robust immunity and heterologous protection against influenza in mice elicited by a novel recombinant NP-M2e fusion protein expressed in E. coli.

BACKGROUND: The 23-amino acid extracellular domain of matrix 2 protein (M2e) and the internal nucleoprotein (NP) of influenza are highly conserved among viruses and thus are promising candidate antigens for the development of a universal influenza vaccine. Various M2e- or NP-based DNA or viral vector vaccines have been shown to have high immunogenicity; however, high cost, complicated immunization procedures, and vector-specific antibody responses have restricted their applications. Immunization with an NP-M2e fusion protein expressed in Escherichia coli may represent an alternative strategy for the development of a universal influenza vaccine. METHODOLOGY/PRINCIPAL FINDINGS: cDNA encoding M2e was fused to the 3' end of NP cDNA from influenza virus A/Beijing/30/95 (H3N2). The fusion protein (NM2e) was expressed in E. coli and isolated with 90% purity. Mice were immunized with recombinant NM2e protein along with aluminum hydroxide gel and/or CpG as adjuvant. NM2e plus aluminum hydroxide gel almost completely protected the mice against a lethal (20 LD(50)) challenge of heterologous influenza virus A/PR/8/34. CONCLUSIONS/SIGNIFICANCE: The NM2e fusion protein expressed in E. coli was highly immunogenic in mice. Immunization with NM2e formulated with aluminum hydroxide gel protected mice against a lethal dose of a heterologous influenza virus. Vaccination with recombinant NM2e fusion protein is a promising strategy for the development of a universal influenza vaccine.

[Peptide mapping of H-2d restricted T-cell epitope against six antigens of HIV-1 subtype B'/C by ELISPOT assay].

The purpose is to screen and identify the specific H-2d restricted T-cell epitopes. These epitopes are used to investigate the cellular immune response of BALB/c (H-2d) mice immunized with a HIV-1 vaccine which expresses six antigens of gp160, gag, pol, rev, tat and nef of HIV subtype B'/C. A replicating DNA vaccine and a non-replicating recombinant vaccinia virus vector, both expressing the six antigens mentioned above, were used to immune BALB/c (H-2d) mice in a prime-boost regiment. The six peptide libraries of HIV B'/C corresponding respectively to the six complete antigens were pooled according to a designed matrix format and used to test for IFN-gamma production from splenocytes of immunized mice by an enzyme-linked immunospot (IFN-gamma ELISPOT) assay. The ELISPOT data indicated that two of seven Gag-specific T-cell epitope peptides were identified to be the novel epitopes. One of three Pol-specific T-cell epitope is unreported. One novel epitope was confirmed in two gp160-specific T-cell epitope peptides. One Nef-specific T-cell epitope was identified. Three Tat-specific T-cell epitope peptides were continuous sequences in Tat peptide library and all contained either complete or partial sequence reported. Rev-specific T-cell epitope was not be found. The specific T-cell epitopes (H-2d restricted) were identified by IFN-7 ELISPOT assay, which could be used to detect the cellular immune response of BALB/c mice immunized with the HIV-1 vaccine expressing six antigens of gp160, gag, pol, rev, tat and nef of HIV subtype B'/C.

[The non-replicating recombinant vaccinia virus expressing six genes of HIV-1 can be passaged stably in CEF].

To investigate the genetic stability (including the vector of vaccinia virus and six foreign genes: gp160, gag, pol, rev, tat and nef) of the HIV-1 non-replicating recombinant vaccinia virus (rNTV-C). rNTV-C was serially passaged to passage 25 (P25) in primary chicken embryo fibroblast (CEF). P9, P12, P15 and P25 were selected to study the genetic stability in four aspects, including the genetic stability of viral vector, the genetic stability of six foreign genes, the expressing stability of foreign genes and the genetic loss of foreign genes. The results showed that the viral vector was non-replicated vaccinia virus of Tiantan strain and was passaged stably; foreign gene sequences matched with designed sequences, the insert sites were right, and the nucleotide mutation rate was less than one over ten thousands within different passages of rNTV-C; the target proteins could be expressed effectively, and the expression level was stable within different passages of rNTV-C; the genetic loss of gag and nef was less than 5% within different passages of rNTV-C. The above results provided important data for the vaccine production.

IL-15 increases the frequency of effector memory CD8+ T cells in rhesus monkeys immunized with HIV vaccine.

Several studies have suggested that interleukin (IL)-15 is a promising adjuvant that promotes cellular immunity when administered with human immunodeficiency virus (HIV) vaccine. Here we evaluated the effect of IL-15 plasmid on HIV-specific immune responses, especially cellular immunity, in eight rhesus monkeys. These monkeys were immunized three times with HIV DNA vaccine with or without IL-15 plasmid and boosted with recombinant Tiantan strain vaccinia virus-based HIV vaccine (rTV) 22 weeks after the first immunization. Although we did not detect any significant differences in the HIV-specific CD8(+) T-cell response between monkeys with IL-15 coimmunization and monkeys with HIV vaccine alone, our results showed that the frequency of effector CD8(+) memory T cells in the peripheral blood was significantly higher in monkeys with IL-15 coimmunization than those with HIV vaccine alone at almost all of the time points examined. Furthermore, the titers of anti-HIV antibodies were higher in Group T than those in Group C after rTV boosting. These findings in rhesus monkeys suggest that IL-15 may be useful as a cytokine adjuvant for HIV vaccine.

[Expression and immunity of multi-HIV B'/C subype genes in replicating DNA vaccines].

To understand the effect of various gene structures of HIV B'/C subtype on the gene expression and immunity in DNA vaccine, replicating DNA vector pSCK2 was used to construct seven DNA vaccines carrying one or more of HIV B'/C subtype genes: gagpol, gp160 and rtn (rev, tat and nef fusion gene). Immunofluorescence staining indicated that Gag, Gp160, Rev, Tat and Nef could be expressed from the seven DNA vaccines. Stronger expression was observed with the gene in single-gene expression plasmid or with the gene located at upper-IRES in double- or multi-gene expression plasmid. ELISA test showed that Gag induced higher antibody response, but the antibody titers stimulated by Gp160, Pol, or RTN were very low. Both Gag single-gene expression plasmid and Gag-RTN double-gene expression plasmid separately inoculating induced stronger antibody response against Gag than Gag-Gp160 double-gene expression plasmid and Gagpol-Gp160-RTN multi-gene expression plasmid or combined inoculation of Gag and Gp160 single-gene expression plasmids did. ELISPOT detection showed that all the seven DNA vaccines could stimulate cellular immune response against Gag, Pol, Gp160, Tat, and Nef, respectively. Gagpol or Gp160 single-gene expression plasmid separately inoculating stimulated the strongest cellular immune response. Tat and Nef expressed in all the plasmids induced similar immune response. These results indicated that HIV B'/C subtype genes gagpol, gp160 and rtn could be efficiently expressed in the replicating DNA vaccine vector, single-gene expression plasmid had the higher gene expression level and induced stronger immune response; combined immunization of Gagpol and Gp160 had dramatically lower immunity than Gagpol or Gp160 separated immunization did. Immunity of RTN had no difference between combined and separated immunizations. Therefore, in case of immunization with DNA vaccines containing different HIV genes, it is necessary to optimize the combined immunization procedure, especially for the combination of Gag and Gp160-containing vaccines.

[Vaccination with three HIV-1 cross neutralizing epitopes fused to HBV S antigen could induce robust antibody immune response in mice].

To enhance immunogenicity of HIV-1 cross neutralizing epitopes , three HIV-1 cross neutralizing epitopes (ELDKWA, NWFDIT, GPGRAFY) were fused to 3' end of HBV S gene by PCR cloning technology, respectively. Three vaccinia virus (Tiantan strain) recombinants expressing separately the three fusion genes were subsequently constructed, named as RVJ1175S-2F5 (ELDKWA), RVJ1175S-4E10 (NWFDIT) and RVJ1175S-447-52D (GPGRAFY), respectively. From the supernatants of CEF cells infected by these vaccinia recombinants, three subunit vaccines (PS-2F5, PS-4E10 and PS-447-52D) were prepared after purification. Biology and immunology characteristics of these fusion antigens in vaccinia recombinants and subunit vaccines were comparatively studied. It was confirmed by PCR and sequencing that the fusion genes were inserted into the TK locus of vaccinia virus (Tiantan strain) correctly. The Fusion proteins were expressed efficiently and secreted into supernatant of the infected cells, which was demonstrated by HBsAg ELISA test. Two typical HBsAg bands of 23kD and 27kD were detected in all the purified samples by SDS-PAGE. These two bands were reacted well to HBsAb and corresponding HIV-1 monoclonal antibodies 2F5, 4E10 and 447-52D. BALB/c mice were immunized with subunit and vaccinia recombinant vaccines by intraperitoneal injection. High levels of HBsAb and anti-HIV-1 cross neutralizing epitope antibody in peripheral blood of immunized mice were tested by ELISA, and all the antibody titers induced by three subunit vaccines were higher than that induced by correlated vaccinia recombinants in mice. This work provides a basis for future study on neutralizing activity of these immunized sera and enhancing immune effect through the combined immunization with different type of vaccines.