Biomedical subjects

V Durmanová

Publications and source records attributed to V Durmanová.

8 recordsLinked to original sources

PubMed2006

Developments in herpes simplex virus vaccines: old problems and new challenges.

Vaccination has remained the best method for preventing virus spread. The herpes simplex virus (HSV) candidate vaccines tested till now were mostly purified subunit vaccines and/or recombinant envelope glycoproteins (such as gB and gD). In many experiments performed in mice, guinea pigs and rabbits, clear-cut protection against acute virus challenge was demonstrated along with the reduction of the extent of latency, when established in the immunized host. The immunotherapeutic effect of herpes vaccines seems less convincing. However, introduction of new adjuvants, which shift the cytokine production of helper T-cells toward stimulation of cytotoxic T-cells (TH1 type cytokine response), reveals a promising development. Mathematical analysis proved that overall prophylactic vaccination of seronegative women, even when eliciting 40-60 % antibody response only, would reduce the frequency of genital herpes within the vaccinated population. Even when partially effective, immunotherapeutic vaccination might represent a suitable alternative of chronic chemotherapy in recurrent labial and genital herpes.

Adjuvants, Immunologic↗

PubMed2004

Pathogenetical characterization of isolate MHV-60 of mouse herpesvirus strain 68.

Infection of mice with mouse herpesvirus strain 68 (MHV-68) is an excellent small animal model of gammaherpesvirus pathogenesis in a natural host. We carried out comparative studies on MHV-60, another isolate of MHV-68. The acute infection of BALB/c mice inoculated intranasally (i.n.) with MHV-60 as well as its impact on tumor development were investigated. During the acute phase of infection the lungs were the main tissues infected. Our results show that MHV-60 has similar pathological features like other 4 isolates so far examined, namely MHV-72, MHV-78, MHV-Sumava inclusive of MHV-68. Nevertheless, MHV-60 differed from other isolates in following features: (i) the acute phase of infection was established very soon and lasted 10 days post infection (p.i.) in contrast to 14-28 days p.i. in the abovementioned isolates with a peak on days 3-5 p.i. The virus could also be recovered from the spleen, thymus and kidneys but not in other investigated organs. A lymphoproliferative response was associated with splenomegaly. At this time an increase in the number of leukocytes and appearance of atypical leukocytes in peripheral blood were observed. (ii) the infection was localized in the lungs and spleen, while in other isolates it was detected in a much broader scale of organs, and (iii) the acute phase of infection was accompanied by a massive splenomegaly, which was characteristic for the chronic phase of infection. Despite the fact that after clearance of the acute infection the virus was hardly detected, the tumor formation was later observed in 22% of infected mice as compared to 5% in control non-infected mice.

Animals↗

PubMed2004

Expression of herpes simplex virus 1 glycoprotein D in prokaryotic and eukaryotic cells.

Recombinant plasmids encoding either the full-length glycoprotein D (FLgD) or truncated gDs were constructed. The recombinant plasmids were expressed in Escherichia coli and BHK-21 cells. The strongest expression was obtained with the recombinant plasmid encoding a truncated gD which corresponded to the gD ectodomain. The cells transformed with this plasmid showed good exponential growth ensuring satisfactory yields of the expressed polypeptide in the form of the fusion protein. The fusion protein was biotinylated and efficiently purified. The shortest truncated gD, which contained the main continuous antigenic locus VII binding neutralization antibody and additional continuous antibody binding epitopes, still reacted with specific antibody as proven by immunoblot analysis. In addition, a shuttle vector for expression of FLgD in mammalian cells was constructed. This vector-transfected BHK-21 cells expressed gD for 40 days during 9 consecutive passages. The expression of gD began on day 2 and culminated at day 9 post transfection (p.t.).

Animals↗

PubMed2003

Detection of immediate early protein ICP27/IE63 and thymidine kinase in the course of reactivation of latent herpes simplex virus 1 infection.

We followed the kinetics of reactivation of latent Herpes simplex virus 1 (HSV-1) infection established in rabbits by corneal route. The corresponding trigeminal ganglia (TG) were cultured and the culture medium was examined at daily intervals for release of infectious virus. Sections from the cultured TG fragments were stained with antisera against non-structural proteins such as the immediate early (IE) protein ICP27 and the early (E) proteins thymidine kinase (TK), the large subunit of ribonucleotide reductase (RR1), the ori-binding protein OBP and with a human serum obtained from volunteers immunized with an experimental subunit HSV-1 envelope (env) vaccine containing late structural proteins gB1, gC1, gD1 and gG1 (env antiserum). By indirect immunofluorescence (IF) test, ICP27 was detected in a few neurons from day 1 post explantation (p.e.), while TK was observed in neurons from day 2 p.e. Fluorescence with the human env antiserum was seen at day 3 p.e. The RR1 and OBP antisera stained productively infected Vero cells from 3 and 4 hrs post inoculation (p.i.), respectively. However, these sera showed no IF in cultured ganglion fragments at any interval examined. Our results showed the same cascade of HSV-1 IE and E protein expression during productive infection and reactivation in vitro.

Animals↗

PubMed2001

A simple procedure for expression and purification of selected non-structural (alpha and beta) herpes simplex virus 1 (HSV-1) proteins.

The expression and isolation of herpes simplex virus 1 (HSV-1) immediate early (alpha) IE63 (ICP27) and of the early (beta) thymidine kinase (Tk) polypeptides in Escherichia coli JM 109 cells transformed with the PinPoint Xa-1 (Promega) plasmid construct carrying either the HSV-1 UL54 or UL23 genes are described. The resulting biotinylated fusion protein(s) could be easily induced and were purified in appropriate amounts by means of a monomeric avidin-conjugated resin (SoftLink Soft Release Avidin Resin, Promega) provided that: (1) the exponential growth of the selected transformed cells was monitored carefully; (2) the post-induction harvest interval was properly chosen; and (3) the period for adsorption to the avidin resin suitably adjusted. The isolated protein(s), although partially digested in the case of the IE63 polypeptide, were suitable antigen(s) for immunization of various animal species. Co-purification of trace amounts of endogenous biotinylated protein(s) produced in E. coli was eliminated by shortening the duration of adsorption to the avidin resin.

Electrophoresis, Polyacrylamide Gel↗

PubMed2001

The UL9 ori-binding protein of herpes simplex virus 1: its expression and localization in vero cells.

The ori-binding protein (OBP), an early protein which is encoded by the herpes simples virus 1 (HSV-1) UL9 gene and initiates the replication of viral DNA, was expressed in Escherichia coli, purified on an avidin resin and used for preparation of a mouse antiserum to OBP (OBP antiserum). Expression and localization of OBP in HSV-1-infected Vero cells was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and indirect immunofluorescence test. RT-PCR revealed the presence of abundant UL9 transcripts from 3 to 12 hrs post infection (p.i). Traces of UL9 mRNA were detected already at 1.5 hr p.i. The OBP antiserum detected clumps of irregularly shaped structures in the nuclei of infected Vero cells first at 4 hrs p.i. These nuclear structures peaked at 5-6 hrs p.i. and later on (at 8-12 hrs p.i.) they changed into fine granules filling the whole nucleus.

Animals↗

PubMed2000

Early expression of herpes simplex virus (HSV) proteins and reactivation of latent infection.

During the last decade, new data accumulated describing the early events during herpes simplex virus 1 (HSV-1) replication occurring before capsid formation and virion envelopment. The HSV virion carries its own specific transcription initiation factor (alpha-TIF), which functions together with other components of the cellular transcriptase complex to mediate virus-specific immediate early (IE) transcription. The virus-coded IE proteins are the transactivator and regulatory elements modulating early transcription and subsequent translation of nonstructural virus-coded proteins needed mainly for viral DNA synthesis and for the supply of corresponding nucleoside components. They also cooperate at the late transcription and translation of the virion (capsid, tegument and envelope) proteins. In addition, the transactivator IE proteins down-regulate their own transcription, while others facilitate viral mRNA processing or interfere with the presentation of newly synthesized virus antigens. Establishment of latency is closely related to the transcription of a separate category of transcripts, termed latency-associated (LAT). Formation of LATs occurs mainly in nondividing neurons which are metabolically less active and express lower levels of cellular transcription factors (nonpermissive cells). Expression of the stable non-spliced (2 kb), and especially of stable spliced (1.5 and 1.45 kb) LATs is a prerequisite for HSV reactivation. Different HSV genomes (from various HSV strains) do not undergo IE transcription at the same rate. Restricted IE transcription and the absence of viral DNA synthesis favors LAT formation and persistence of the silenced genome. Uneven levels of LAT expression and differences in the metabolic state of carrier neurons influence the reactivation competence. Under artificial or natural activation conditions, sufficient amounts of IE transactivator proteins and proteins promoting nucleoside metabolism are synthesized even in the absence of the viral alpha-TIF facilitating reactivation.

Animals↗

PubMed1999

Increased neoplasm development due to immunosuppressive treatment with FK-506 in BALB/C mice persistently infected with the mouse herpesvirus (MHV-72).

BALB/c mice were infected with the lymphotropic mouse gammaherpesvirus (MHV-72). At late (7-12 months) post-infection intervals the latent virus was detected in the cells of lymphatic system (peripheral blood, lymphocytes and macrophages, thymocytes, lymph nodes, bone marrow, and peritoneal macrophages,) and in the spleen, lungs, liver, and kidney by cocultivation as well as by explantation. The MHV-72 infected mice, in which latency had been established, were treated with the immunosuppressive (IS) drug FK-506 (2 mg/kg/mouse for 30 days). This treatment increased the probability of virus reactivation by over two-fold. During the post-treatment observation period of 19 months, the incidence of lymphomas and the development of MHV-related lymphoproliferative and hemoblastic disorders raised to nearly five-fold in the drug treated mice as compared to untreated animals.

Animals↗