Correction: A vesicular stomatitis virus-based prime-boost vaccination strategy induces potent and protective neutralizing antibodies against SARS-CoV-2.

[This corrects the article DOI: 10.1371/journal.ppat.1010092.].

A vesicular stomatitis virus-based prime-boost vaccination strategy induces potent and protective neutralizing antibodies against SARS-CoV-2.

The development of safe and effective vaccines to prevent SARS-CoV-2 infections remains an urgent priority worldwide. We have used a recombinant vesicular stomatitis virus (rVSV)-based prime-boost immunization strategy to develop an effective COVID-19 vaccine candidate. We have constructed VSV genomes carrying exogenous genes resulting in the production of avirulent rVSV carrying the full-length spike protein (SF), the S1 subunit, or the receptor-binding domain (RBD) plus envelope (E) protein of SARS-CoV-2. Adding the honeybee melittin signal peptide (msp) to the N-terminus enhanced the protein expression, and adding the VSV G protein transmembrane domain and the cytoplasmic tail (Gtc) enhanced protein incorporation into pseudotype VSV. All rVSVs expressed three different forms of SARS-CoV-2 spike proteins, but chimeras with VSV-Gtc demonstrated the highest rVSV-associated expression. In immunized mice, rVSV with chimeric S protein-Gtc derivatives induced the highest level of potent neutralizing antibodies and T cell responses, and rVSV harboring the full-length msp-SF-Gtc proved to be the superior immunogen. More importantly, rVSV-msp-SF-Gtc vaccinated animals were completely protected from a subsequent SARS-CoV-2 challenge. Overall, we have developed an efficient strategy to induce a protective response in SARS-CoV-2 challenged immunized mice. Vaccination with our rVSV-based vector may be an effective solution in the global fight against COVID-19.

Induction of protective immune responses against a lethal Zika virus challenge post-vaccination with a dual serotype of recombinant vesicular stomatitis virus carrying the genetically modified Zika virus E protein gene.

The development of a vaccine to prevent Zika virus (ZIKV) infection has been one of the priorities in infectious disease research in recent years. There have been numerous attempts to develop an effective vaccine against ZIKV. It is imperative to choose the safest and the most effective ZIKV vaccine from all candidate vaccines to control this infection globally. We have employed a dual serotype of prime-boost recombinant vesicular stomatitis virus (VSV) vaccine strategy, to develop a ZIKV vaccine candidate, using a type 1 IFN-receptor knock-out (Ifnar-/-) mouse model for challenge studies. Prime vaccination with an attenuated recombinant VSV Indiana serotype (rVSVInd) carrying a genetically modified ZIKV envelope (E) protein gene followed by boost vaccination with attenuated recombinant VSV New Jersey serotype (rVSVNJ) carrying the same E gene induced robust adaptive immune responses. In particular, rVSV carrying the ZIKV E gene with the honeybee melittin signal peptide (msp) at the N terminus and VSV G protein transmembrane domain and cytoplasmic tail (Gtc) at the C terminus of the E gene induced strong protective immune responses. This vaccine regimen induced highly potent neutralizing antibodies and T cell responses in the absence of an adjuvant and protected Ifnar-/- mice from a lethal dose of the ZIKV challenge.

Myoepithelial carcinoma of tibia mimic giant cell tumor: a case report with emphasis on MR features.

Intraosseous myoepithelial carcinoma is an extremely rare type of bone tumor that most often presents in the long tubular bones, but also occurs in small tubular bones and the axial skeleton. We report the radiographic images and complete magnetic resonance (MR) features of a 44-year-old male with right knee pain of 7 months' duration. The radiographic findings and convention MR images indicated a giant cell tumor of the bone. The dynamic contrast-enhanced images showed a patent with the early wash-in and early wash-out usually noted in a giant cell tumor of the bone. Only water restriction on diffusion-weighted imaging (DWI) showed the malignant impression. Care should be taken when conventional images indicate giant cell tumor of the bone, as intraosseous myoepithelial carcinoma, although rare, can mimic this more common diagnosis. Further studies with DWI are warranted.

First Phase I human clinical trial of a killed whole-HIV-1 vaccine: demonstration of its safety and enhancement of anti-HIV antibody responses.

BACKGROUND: Vaccination with inactivated (killed) whole-virus particles has been used to prevent a wide range of viral diseases. However, for an HIV vaccine this approach has been largely negated due to inherent safety concerns, despite the ability of killed whole-virus vaccines to generate a strong, predominantly antibody-mediated immune response in vivo. HIV-1 Clade B NL4-3 was genetically modified by deleting the nef and vpu genes and substituting the coding sequence for the Env signal peptide with that of honeybee melittin signal peptide to produce a less virulent and more replication efficient virus. This genetically modified virus (gmHIV-1NL4-3) was inactivated and formulated as a killed whole-HIV vaccine, and then used for a Phase I human clinical trial (Trial Registration: Clinical Trials NCT01546818). The gmHIV-1NL4-3 was propagated in the A3.01 human T cell line followed by virus purification and inactivation with aldrithiol-2 and γ-irradiation. Thirty-three HIV-1 positive volunteers receiving cART were recruited for this observer-blinded, placebo-controlled Phase I human clinical trial to assess the safety and immunogenicity. RESULTS: Genetically modified and killed whole-HIV-1 vaccine, SAV001, was well tolerated with no serious adverse events. HIV-1NL4-3-specific PCR showed neither evidence of vaccine virus replication in the vaccine virus-infected human T lymphocytes in vitro nor in the participating volunteers receiving SAV001 vaccine. Furthermore, SAV001 with adjuvant significantly increased the pre-existing antibody response to HIV-1 proteins. Antibodies in the plasma of vaccinees were also found to recognize HIV-1 envelope protein on the surface of infected cells as well as showing an enhancement of broadly neutralizing antibodies inhibiting tier I and II of HIV-1 B, D, and A subtypes. CONCLUSION: The killed whole-HIV vaccine, SAV001, is safe and triggers anti-HIV immune responses. It remains to be determined through an appropriate trial whether this immune response prevents HIV infection.

Creation of matrix protein gene variants of two serotypes of vesicular stomatitis virus as prime-boost vaccine vectors.

UNLABELLED: To take advantage of live recombinant vesicular stomatitis viruses (rVSVs) as vaccine vectors for their high yield and for their induction of strong and long-lasting immune responses, it is necessary to make live vaccine vectors safe for use without losing their immunogenicity. We have generated safer and highly efficient recombinant VSV vaccine vectors by combining the M51R mutation in the M gene of serotype VSV-Indiana (VSVInd) with a temperature-sensitive mutation (tsO23) of the VSVInd Orsay strain. In addition, we have generated two new serotype VSV-New Jersey (VSVNJ) vaccine vectors by combining M48R and M51R mutations with G22E and L110F mutations in the M gene, rVSVNJ(G22E M48R M51R) [rVSVNJ(GMM)] and VSVNJ(G22E M48R M51R L110F) [rVSVNJ(GMML)]. The combined mutations G21E, M51R, and L111F in the M protein of VSVInd significantly reduced the burst size of the virus by up to 10,000-fold at 37°C without affecting the level of protein expression. BHK21 cells and SH-SY5Y human neuroblastoma cells infected with rVSVInd(GML), rVSVNJ(GMM), and rVSVNJ(GMML) showed significantly reduced cytopathic effects in vitro at 37°C, and mice injected with 1 million infectious virus particles of these mutants into the brain showed no neurological dysfunctions or any other adverse effects. In order to increase the stability of the temperature-sensitive mutant, we have replaced the phenylalanine with alanine. This will change all three nucleotides from UUG (leucine) to GCA (alanine). The resulting L111A mutant showed the temperature-sensitive phenotype of rVSVInd(GML) and increased stability. Twenty consecutive passages of rVSVInd(GML) with an L111A mutation did not convert back to leucine (UUG) at position 111 in the M protein gene. IMPORTANCE: Recombinant vesicular stomatitis viruses as live vaccine vectors are very effective in expressing foreign genes and inducing adaptive T cell and B cell immune responses. As with any other live viruses in humans or animals, the use of live rVSVs as vaccine vectors demands the utmost safety. Our strategy to attenuate rVSVInd by utilizing a temperature-sensitive assembly-defective mutation of L111A and combining it with an M51R mutation in the M protein of rVSVInd significantly reduced the pathogenicity of the virus while maintaining highly effective virus production. We believe our new temperature-sensitive M gene mutant of rVSVInd(GML) and M gene mutants of rVSVNJ(GMM) and rVSVNJ(GMML) add excellent vaccine vectors to the pool of live viral vectors.

Monovalent and trivalent VSV-based COVID-19 vaccines elicit neutralizing antibodies and CD8+ T cells against SARS-CoV-2 variants.

Recombinant vesicular stomatitis virus (rVSV) vaccines expressing spike proteins of Wuhan, Beta, and/or Delta variants of SARS-CoV-2 were generated and tested for induction of antibody and T cell immune responses following intramuscular delivery to mice. rVSV-Wuhan and rVSV-Delta vaccines and an rVSV-Trivalent (mixed rVSV-Wuhan, -Beta, -Delta) vaccine elicited potent neutralizing antibodies (nAbs) against live SARS-CoV-2 Wuhan (USAWA1), Beta (B.1.351), Delta (B.1.617.2), and Omicron (B.1.1.529) viruses. Prime-boost vaccination with rVSV-Beta was less effective in this capacity. Heterologous boosting of rVSV-Wuhan with rVSV-Delta induced strong nAb responses against Delta and Omicron viruses, with the rVSV-Trivalent vaccine consistently effective in inducing nAbs against all the SARS-CoV-2 variants tested. All vaccines, including rVSV-Beta, elicited a spike-specific immunodominant CD8+ T cell response. Collectively, rVSV vaccines targeting SARS-CoV-2 variants of concern may be considered in the global fight against COVID-19.

Genetically modified VSV(NJ) vector is capable of accommodating a large foreign gene insert and allows high level gene expression.

It is desirable to develop a RNA virus vector capable of accommodating large foreign genes for high level gene expression. Vesicular stomatitis virus (VSV) has been used as a gene expression vector, especially Indiana serotype (VSV(Ind)), but less with New Jersey serotype (VSV(NJ)). Here, we report constructions of genetically modified rVSV(NJ) vector carrying various lengths of human hepatitis C virus (HCV) non-structural (NS) protein genes, level of inserted gene expression and characterization of rVSV(NJ). We modified the M gene of VSV(NJ) by changing methionine to arginine at positions 48 and 51 (rVSV(NJ)-M) (Kim and Kang, 2007) for construction of rVSV(NJ) with various lengths of HCV non-structural genes. The NS polyprotein genes of HCV were inserted between the G and L genes of the rVSV(NJ)-M vector, and recombinant VSV(NJ)-M viruses with HCV gene inserts were recovered by the reverse genetics. The recombinant VSV(NJ)-M vector with the HCV NS genes express high levels of all different forms of the NS proteins. The electron microscopic examination showed that lengths of recombinant VSV(NJ)-M without gene of interests, VSV(NJ)-M with a gene of HCV NS3 and NS4A (VSV(NJ)-M-NS3/4A), VSV(NJ)-M with a gene of HCV NS4AB plus NS5AB (VSV(NJ)-M-NS4AB/5AB), and VSV(NJ)-M carrying a gene of HCV NS3, NS4AB, and NS5AB (VSV(NJ)-M-NS3/4AB/5AB) were 172±10.5 nm, 201±12.5 nm, 226±12.9 nm, and 247±18.2 nm, respectively. The lengths of recombinant VSVs increased approximately 10nm by insertion of 1kb of foreign genes. The diameter of these recombinant viruses also increased slightly by longer HCV gene inserts. Our results showed that the recombinant VSV(NJ)-M vector can accommodate as much as 6000 bases of the foreign gene. We compared the magnitude of the IFN induction in mouse fibroblast L(Y) cells infected with rVSV(NJ) wild type and rVSV(NJ) M mutant viruses and show that the rVSV(NJ) M mutant virus infection induced a higher level of the IFN-ß compare to the wild type virus. In addition, we showed that the NS protein expression level in IFN-incompetent cells (Mouse-L) infected with rVSV(NJ)-M viruses was higher than in IFN-competent L(Y) cells. In addition, we confirmed that HCV NS protein genes were expressed and properly processed. We also confirmed that NS3 protein expressed from the rVSV(NJ)-M cleaves NS polyprotein at junctions and that NS4A plays an important role as a co-factor for NS3 protease to cleave at the NS4B/5A site and at the NS5A/5B site.

[Characteristics and evolution of hydrochemical compositions of freshwater lake in Tibetan plateau].

Freshwater lake of the Tibetan plateau has great ecological value and extreme vulnerability. The water samples have been collected in Daggyaima Co at an altitude of 5 080 m +/- 10 m. This study has investigated the sources, the major controlling factors of the major ions, and hydrochemical evolution trend according to the hydrochemical composition of water samples. The results showed that the major cations and anion of lake water were Ca(2+), Na(+) and HCO3(-), respectively, and the hydrochemical type was HCO3 -Ca, The total dissolved solid (TDS) was in the range of 71.2-199.8 mg x L(-1). The concentrations of EC, Ca(2+) and HCO3(-) in water samples from Southeast areas were relatively low resulted from the rich aluminum and poor calcium of geological background and the dilution effect of surface runoff. The Na(+)/(Na(+) + Ca(2+)) of water samples was 0.08-0.75, Cl(-) /(Cl(-) + HCO3(-)) was 0.11-0.35, Ca/Na was 0.58, Mg/Ca was 0.12, and HCO3/Na was 1.46, which suggested that the hydrochemical composition of the lake water was mainly controlled by silicate rock weathering based on Gibbs model and analysis of elemental stoichiometry. The minerals participated in weathering processes included plagioclase (anorthite, albite), potassium feldspar, biotite, calcite, dolomite, gypsum, rock salt, etc. The average value of K/Na was 0.059, indicating the weathering level of potassium feldspar was relatively low. The saturation index (SI) of calcite, dolomite, gypsum, quartz and hematite contained in the lake was higher than zero, while the SI of rock salt was lower than 0, revealing the trend that the freshwater lake is turning into salt water lake in Tibetan Plateau.

[Weathering seasonal variations in karst valley in southwest China].

Jialing River is a 1st grade tributary of upstream Yangzi River. In two years, Samples were collected monthly in Wentang Gorge section of Jialing River and analyzed multi-parameters including hydrochemistry and isotopes. Thus, a general result was concluded that the hydrochemical characteristic of Jialing River in Wentang gorge is controlled by weathering of stratum and the hydrochemical type is HCO3(-) -Ca. Most irons were influenced by dilution, which had higher concentrations in dry season than that in rainy season, but nitrate. Nitrate, which was controlled by human activities, has higher concentrations in rainy season. However, some other analyst revealed weathering impacts. The contrast ratio of (Ca(2+) + Mg2+) and HCO3- were between 0.5-1, the same as (Ca(2+) + Mg2+) and (HCO3(-) + SO4(2-)), Which implied that the weathering impacts in this basin was mainly carbonated and sulfate weathering of carbonated, and sulphate rocks weathering was not so significant. The values of delta13C(HCO3- in Jialing River were -8.74 per thousand(-) - 7.36 per thousand, and delta34S(SO)(4)2 - was 14.43 per thousand in dry season and 12.21 per thousand in rainy season. The data of isotopes inferred that, in rainy season sulfate weathering of carbonated and sulphate rocks weathering both had more impacts and sulphate rocks weathering played a more important role than sulfate weathering of carbonated, but, in dry season, carbonated weathering of carbonated was more meaningful.

[Spatial and temporal variability of nitrate contaminant in groundwater in Jinfo Mt. area, Chongqing, China].

The geochemical background of nitrate in groundwater in Jinfo Mt. area was determined, and spatial and temporal variability of nitrate contaminant was analyzed using geochemical, statistical and GIS methods. Twenty-three samples were collected from groundwater discharge points in the study area during 1976-1977, 2004-2006 and 2009, and mass concentration of nitrate in groundwater was tested. The results showed that the geochemical background of nitrate in groundwater in study area was in the range of 0.72-2. 00 mg x L(-1), and the threshold of anomaly was 3.20 mg x L(-1). During 2004-2006 and 2009, the average values of nitrate concentration in groundwater in Jinfo Mt. natural reserve were 2.08, 2.67, 2.59 and 3.92 mg x L(-1); and were 39.08, 25.46, 17.99 and 13.73 mg x L(-1) in the groundwater out of the reserve; the average over-limit rates (standard limit NO3(-) -N < or = 10mg x L(-1)) were 451.64%, 478.61%, 331.85% and 145.67%; the maximum over-limit rates were 1 475.81%, 1 080.39%, 538.20% and 361.78%. Results of interpolation showed that the high value centers of nitrate concentration in groundwater in study area were changing over time, but districts with low nitrate concentration in groundwater in study area were distributed along Jinfo Mt. natural reserve. The application of environmental policy measures and industrial restructuring implemented were reasonable and successful, which had a positive effect to environmental protection.

Expression and processing of human immunodeficiency virus type 1 gp160 using the vesicular stomatitis virus New Jersey serotype vector system.

The Indiana serotype of vesicular stomatitis virus (VSV(IND)), but not the New Jersey serotype (VSV(NJ)), has been widely used as a gene expression vector. In terms of prime-boost-based vaccine strategies, it would be desirable to use two different VSV serotypes to avoid immunity against the priming viral vector. Here, we report that we have applied the VSV(NJ) vector system for expression of the env gene of human immunodeficiency virus type 1 (HIV-1). The HIV-1 env gene was inserted into the VSV(NJ) vector system at two different sites: between the P and M genes (NP-gp160-MGL) and between the G and L genes (NPMG-gp160-L). The HIV-1 env gene product, gp160, was efficiently expressed and processed in cells infected with either of these two recombinant VSV-HIV-1(gp160) viruses. In this study, we have investigated the applicability of the VSV(NJ) vector system for foreign gene expression.

[Characterization of genome of A/Guangzhou/333/99(H9N2) virus].

BACKGROUND: To understand the characterization of genome of a strain of avian influenza A H9N2 virus repeatedly isolated from a child with influenza illness. Thereafter to reveal the origin of this H9N2 virus. METHODS: Viruses were passed in embryonated hen eggs and virion RNA was extracted from allantoic fluid and reverse transcribed to synthesize cDNA. cDNA was amplified by PCR and the PCR product was purified with a purification kit. Afterwards RNA sequence analysis was performed by dideoxynucleotide chain termination and a cloning method. Finally, phylogenetic analysis of the sequencing data was performed with MegAlign (Version 1.03) and Editseg (Version 3.69) softwares. RESULTS: Genome of A/Guangzhou/333/99 (H9N2) virus was closely related to avian influenza A H9N2 virus, but obvious difference from that of A/Duck/Hong Kong/Y439/97(H9N2) virus, as well as its genome did not include any RNA segment derived from human influenza A virus. However, the genes encoding the HA,NA,NP and NS proteins of A/Guangzhou/333/99 virus were derived from those of G9 lineage virus, the rest genes encoding the M and three polymerase (PB2,PB1 and PA) proteins were derived from G1 lineage strain. CONCLUSIONS: A/Guangzhou/333/99 virus was a reassortant derived from reassortment betweenG9 and G1 lineages of avian influenzaA(H9N2) viruses. Therefore, the most possibility is that it is derived from avian influenza A virus directly. The results do not only demonstrate that avian influenza A (H9N2) virus could infect men, but also firstly prove that the genetic reassortment could be occurred between different genetic lineages of avian influenza A (H9N2) viruses in the nature.