Strategies of rational and structure-driven vaccine design for Arenaviruses.

The COVID-19 outbreak has highlighted the importance of pandemic preparedness for the prevention of future health crises. One virus family with high pandemic potential are Arenaviruses, which have been detected almost worldwide, particularly in Africa and the Americas. These viruses are highly understudied and many questions regarding their structure, replication and tropism remain unanswered, making the design of an efficacious and molecularly-defined vaccine challenging.

Eukaryotic transcriptomics in silico: optimizing cDNA-AFLP efficiency.

Background: Complementary-DNA based amplified fragment length polymorphism (cDNA-AFLP) is a commonly used tool for assessing the genetic regulation of traits through the correlation of trait expression with cDNA expression profiles. In spite of the frequent application of this method, studies on the optimization of the cDNA-AFLP assay design are rare and have typically been taxonomically restricted. Here, we model cDNA-AFLPs on all 92 eukaryotic species for which cDNA pools are currently available, using all combinations of eight restriction enzymes standard in cDNA-AFLP screens.

Rapid detection of single nucleotide polymorphisms associated with spinal muscular atrophy by use of a reusable fibre-optic biosensor.

Rapid (<2 min) and quantitative genotyping for single nucleotide polymorphisms (SNPs) associated with spinal muscular atrophy was done using a reusable (approximately 80 cycles of application) fibre-optic biosensor over a clinically relevant range (0-4 gene copies). Sensors were functionalized with oligonucleotide probes immobilized at high density (approximately 7 pmol/cm2) to impart enhanced selectivity for SNP discrimination and used in a total internal reflection fluorescence detection motif to detect 202 bp PCR amplicons from patient samples. Real-time detection may be done over a range of ionic strength conditions (0.

Influences of non-selective interactions of nucleic acids on response rates of nucleic acid fiber optic biosensors.

The immobilization of oligonucleotides to solid surfaces can provide a platform of chemistry that is suitable for the development of biosensor and microarray technologies. Experiments were performed using a fiber optic nucleic acid biosensor based on total internal reflection fluorescence to examine the effects of the presence of non-complementary DNA on the detection of hybridization of complementary target DNA. The work has focused on the rates and extent of hybridization in the presence and absence of non-selective adsorption using fluorescein-labeled DNA.

A novel phosphoramidite method for automated synthesis of oligonucleotides on glass supports for biosensor development.

Two protocols for functionalization of glass supports with hexaethylene glycol (HEG)-linked oligonucleotides were developed. The first method (standard amidite protocol) made use of the 2-cyanoethyl-phosphoramidite derivative of 4,4'-dimethoxytrityl-protected HEG. This was first coupled to the support by standard solid-phase phosphoramidite chemistry followed by extension with a thymidylic acid icosanucleotide.

Cell death in the human leukemia cell line, K-562, induced by antiserum and monoclonal antibodies.

Rabbit polyclonal antiserum, or derived gamma globulin, to K-562 cells induces decreased TdR uptake within hours and cell death without cytolysis in 2-4 days. A panel of nine mAb, reactive with K-562 cells, was grouped on the basis of no effect on growth or TdR uptake, increased uptake, or decreased uptake. Treatment of cells with antiserum, gamma globulin, or mAb of the last group caused single-strand, but not double-strand, DNA fragmentation at a time when the cells were still viable.

Monoclonal antibodies that cross-react with the E1 glycoprotein of different alphavirus serogroups: characterization including passive protection in vivo.

A panel of four monoclonal antibodies (mAb) were produced that cross-react with representatives of two different togavirus serogroups, namely sindbis (SIN) and Semliki Forest (SF) viruses, by ELISA and ADCMC assays. Three of these mAb, IgG2a and IgG2b isotypes, passively protected C3H/Hej mice against 10 and 100 LD50 of SF challenge and one, IgM, did not protect against either challenge dose, or even at 1 LD50. All these mAb were cross-reactive with the E1 glycoprotein of the viruses by immunoblotting in which three different patterns of reactivity were evident, suggesting that three epitopes were involved.

Involvement of cytoplasmic membranes in the non-lytic infection of K-562 cells by Semliki Forest virus.

An analysis of the human leukemia cell line, K-562, infected with Semliki Forest virus, has been made with transmission electron microscopy. In contrast to the usual surface budding of the enveloped virus on the plasma membrane of vertebrate cells leading to cytolysis within 20 h, K-562 cells do not show surface budding, and the cells remain intact for periods of several months. Several unusual features of the infection include: 1) the rough endoplasmic reticulum arranges early into continuous perinuclear chains; 2) during the time of virus replication and release, the nucleocapsids aggregate on the cytoplasmic side of internal vesicles in the region of the cell where the Golgi complex is normally located; and 3) during this same time period, the vesicles are seen to contain enveloped virions and rod-like formations, a result suggesting that budding has occurred into these vesicles.

Peritoneal barrier to the spread of Semliki forest virus in mice.

The LD50 for encephalitis caused by Semliki forest virus in 6- to 8-week-old mice is 1 plaque-forming unit (PFU) in C3H/Ten strain of mice when injected intracerebrally, iv, or in the footpad; however, the LD50 by the ip route is 4 x 10(3) PFU. In the ICR strain of mice at the same age, the LD50 for the intracerebral route is 1 PFU, 10(3) PFU for the iv and footpad routes, and 4 x 10(3) PFU for the ip route. A number of in vivo and in vitro experiments were done to explain the relative resistance to Semliki forest virus injection by the ip route.

Infection of a human leukemia K-562 cell line with Semliki Forest virus.

Infection of the human leukemia hematopoietic stem cell line, K-562, with Semliki Forest virus (SFV) can be characterized by three stages: (1) an early virus-proliferating stage lasting 1 to 4 days post-infection (pi) in which infectious virus is produced in high titers (10(3)pfu/cell) but in which there is minimal cytopathic effect. All cells appear viable by trypan blue dye exclusion, although they do not proliferate, and DNA and cell protein synthesis decrease to less than 3% of uninfected controls within 24 hours; (2) an intermediate stage extending from day 5 to about day 24-30 pi in which the amount of infectious virus declines to low levels. During this stage, viral protein synthesis decreases to undetectable levels, although viral gylcoproteins are readily demonstrated by immunofluorescence and by immunoblot; however, capsid protein appears to degrade within 21 days pi.

Lowered susceptibility of K-562 cells treated with gamma interferon in serum-free medium to natural killer cell-mediated cytolysis.

K-562 cells grown in serum-free medium were treated with gamma interferon (IFN-gamma) and they became significantly less susceptible to natural killer (NK) cell-mediated cytolysis. To examine if this loss in susceptibility was related to induced differentiation events, the presence of various antigens was determined after induction. There was a coincident expression of class I HLA common antigen, although it is not clear if this is a direct causal relationship.

Interferon-gamma-induced expression of class I HLA antigens on K-562 cells grown in serum-free medium.

Human interferon-gamma (IFN-gamma), encoded in Escherichia coli by recombinant human DNA, induces the expression of HLA antigens in the pluripotent hematopoietic stem cell line K-562 grown in serum-free growth medium. Expression was noted in 90% of the cells within 4 days and there was a high density of expression per cell, as determined by cytofluorography. Upon subculture, the cells rapidly lose their ability to express HLA, indicating that the continued presence of IFN-gamma is necessary for the expression.

Continuous or modulation expression of hematopoietic cell antigens in sublines of the leukemia cell line, K-562.

K-562 is described as a pluripotent leukemic cell line which expresses a diversity of hematopoietic cell differentiation antigens. With the use of monoclonal antibodies (MoAb), we show that the expression of these antigens is either "modulated", i.e.

Passive protection across subgroups of alphaviruses by hyperimmune non-cross-neutralizing anti-Sindbis serum.

Extended hyperimmunization of rabbits with Sindbis (SIN) or Semliki Forest (SF) viruses causes the production of antisera that are cross-reactive with virus-infected cells in antibody-dependent, complement-mediated cytotoxicity assays but that do not cross-neutralize viruses in vitro. C3H/HeJ mice given gamma globulin fractionated from the extended hyperimmune antiserum against SIN, but not control sera, were protected from challenge by 100 LD50 of SF, a virus which is in a different subgroup than SIN. All mice survived if the gamma globulin was given 24 hr before challenge virus and partial protection occurred if the globulin was given 24 hr after the virus.

Diversity of cell surface hematopoietic antigens on K-562 sublines identified with monoclonal antibodies.

The surface antigen profile of 8 sublines of K-562 cells, the original line, and the clone RA6 was determined with a panel of 12 monoclonal antibodies reactive with hematopoietic cell differentiation antigens. Cells from all sublines expressed the precursor hematopoietic antigen reactive with RFB-1, the T-cell antigen reactive with OKT17, the B cell/granulocyte antigen reactive with BA-1, the My-1 antigen, and glycophorin A which reacted with R10. A low percentage of cells in some of the sublines expressed platelet/monocyte glycoprotein I binding AN51, monocyte antigen binding 63D3, and an erythroblast/monocyte/platelet antigen binding 5F1.

Cross-reactive target antigen in cell-mediated cytolysis of cells infected with a temperature-sensitive mutant of Sindbis virus.

Cytotoxic T lymphocytes (CTL) against alphavirus-infected L929 cells were generated in mice by two in vivo immunizations of one virus or by in vivo immunization followed by in vitro stimulation of splenocytes with infected peritoneal exudate cells or splenocytes. These CTL caused specific H-2-restricted cytolysis equally well with homologous, heterologous or Sindbis virus ts20 mutant-infected cells. Thus, specific CTL appear to be cross-reactive components in normal immunity to alphaviruses and the E1 glycoprotein is implicated as the target antigen.

Natural killer cell resistance in K-562 cell sublines.

Sublines of the hematopoietic stem cell line K-562 were tested for their susceptibility to human natural killer (NK) cell activity. A correlation was found between the degree of NK-mediated lysis and the presence or absence of particular chromosomal markers. K-562 subline susceptible to lysis by NK were found to express karyotypically a deletion 9- and a marker 8(t1-18), whereas resistant sublines did not express these markers.

Characterization of the Fc(IgG) receptor on the pluripotential leukaemia cell K-562.

K-562 cells express an Fc receptor that is murine isotype IgG specific. The receptor was defined by rosette formation using sheep erythrocytes sensitized with murine monoclonal haemagglutinin (EA) of known isotype. Optimal rosette formation occurred at 4 degrees C or ambient temperature; however, the number of rosettes formed at ambient temperature decreased after 8 h whereas formed rosettes were stable at 4 degrees C.

Detection of immunologically cross-reacting capsid protein of alphaviruses on the surfaces of infected L929 cells.

Hyperimmune, but not normal immune, monospecific antiserum made to capsid protein of Sindbis virus (SIN) was found to cause cytolysis equally well of both SIN- and Semliki Forest virus-infected L929 cells in antibody-dependent, complement-mediated cytotoxicity assays. The cell surface reactivity of the hyperimmune antiserum was also demonstrated by solid-phase radioimmune assays with unfixed infected cells or infected cells fixed with low concentrations of glutaraldehyde (0.025%) before reactivity with antisera.

Identification of immunologically cross-reactive proteins of Sindbis virus: evidence for unique conformation of E1 glycoprotein from infected cells.

Hyperimmune antisera to purified Sindbis (SIN) or Semliki Forest (SF) virus were used to identify alphavirus-specific and cross-reactive proteins in virions and infected cells. The hyperimmune sera participated in homologous and cross-cytolysis of alphavirus-infected cells, and the use of monospecific antisera to SIN structural proteins suggested that E1 and E2 could serve as target proteins in cytolysis. Proteins from purified virions or infected cells were extracted with Nonidet P-40, denatured by procedures for sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose solid supports, and reacted with hyperimmune sera and 125I-labeled protein A (immunoblotting on denatured proteins).

Proliferation of human malignant hematopoietic cells in immunodeficient mice: suppression by antibody to pluripotent K-562 leukemia cells involves direct cytolysis and effector cells.

Six human hematopoetic cell lines were successfully heterotransplanted into athymic (nude) and asplenic-athymic (lasat) neonatal mice. The tumors arising from leukemia and lymphoma cells could then be serially transplanted into adult nude mice. Seven days after the fourth serial mouse passage, each mouse was treated with goat immune gamma globulin against K-562 cells.

Relationship between the Fc receptor for IgG and the specific associated antigen of the pluripotential leukaemia cell line, K-562.

The cells in the K-562 line have been shown to express Fc receptors as demonstrated by rosette formation with sheep erythrocytes (E) sensitized with haemagglutinin (EA). Rosette formation is inhibited by prior incubation of the cells with goat or monkey anti-K-562 serum, gammaglobulin fraction, or Fab fragments. Alkaline aggregated human IgG also inhibits rosette formation.

Antibody lysis of human hematopoietic cells in the absence of complement and effector cells.

The incubation of K-562 leukemia cells with specific goat immunoglobulin resulted in gradual cytolysis in the absence of complement and effector cells. Optimal lysis (100%), reached in 3-4 days, occurred when the concentration of the antibody in the culture medium was about 20 micrograms/ml containing an initial inoculum of 15,000 cells. A lower concentration (10 micrograms/15,000 cells/ml) led to partial cytolysis; the surviving cells, cultured in fresh medium, were still vulnerable to antibody lysis (30 micrograms/ml), thus indicating the lack of an apparent resistant cell population.