Gerald A. Beltz

Inhibition of HeLa cell protein synthesis during adenovirus infection. Restriction of cellular messenger RNA sequences to the nucleus.

The mechanism by which adenovirus type 2 inhibits HeLa cell protein synthesis has been investigated by a quantitative analysis of the synthesis and metabolism of cellular and viral hnRNA† sequences. By the time labeling of cellular proteins is maximally inhibited, 16 hours following adenovirus infection under the conditions we have used, all of the newly made messenger RNA sequences reaching the cytoplasm are viral-specific. Thus, the gradual shift from translation of cellular to translation of viral mRNA species that begins following entry of adenovirus-infected cells into the late phase of the productive cycle, can probably be explained simply by competition between the two types of mRNA for available ribosomes. In order to investigate the mechanism(s) by which adenovirus infection exerts this dramatic change in the biogenesis of HeLa cellular mRNA sequences, the hnRNA fraction has been isolated quantitatively from infected cells after pulselabeling or a pulse-chase. Cellular and viral transcripts were then distinguished by hybridization to viral DNA. The results of these experiments reveal that the rate of synthesis of cellular hnRNA sequences remains constant throughout the course of adenovirus type 2 infection. Moreover, poly(A) is apparently added normally to cellular hnRNA sequences synthesized during the late phase of productive adenovirus infection. This newly synthesized, poly(A)-containing hnRNA cannot be distinguished from the corresponding fraction isolated from mockinfected cells by hybridization to cDNA prepared using uninfected HeLa cell, cytoplasmic, poly(A)-containing RNA as template: it also appears to be metabolized with kinetics similar to those observed in uninfected cells.

Genetically-engineered subunit vaccine against feline leukaemia virus: protective immune response in cats

A recombinant retroviral subunit vaccine has been developed that successfully protects cats from infectious feline leukaemia virus (FeLV) challenge. The antigen used is a non-glycosylated protein derived from the envelope glycoprotein of FeLV subgroup A, expressed in Escherichia coli. This recombinant protein, rgp70D, includes the entire exterior envelope protein gp70, plus the first 34 amino acids from the transmembrane protein p15E. The vaccine consists of purified rgp70D absorbed on to aluminium hydroxide and used in conjunction with a novel saponin adjuvant. Cats immunized with this formulation developed a strong humoral immune response, including neutralizing and feline oncornavirus-associated cell membrane antigen antibodies. Vaccinated animals showed an anamnestic response upon intraperitoneal challenge with FeLV-A, and were protected from viral infection. In contrast, the control animals developed viraemia shortly after the challenge, which in most cases became chronic. Formulation of the same antigen with other widely used adjuvants elicited poor protective immune responses in cats.

Diagnosis of Human Immunodeficiency Virus Infection by Immunoassay Using a Molecularly Cloned and Expressed Virus Envelope Polypeptide: Comparison to Western Blot on 2707 Consecutive Serum Samples

To detect human immunodeficiency virus (HIV) antibodies in a simple enzyme-linked immunoassay (CBre3-EIA), we used an Escherichia coli-expressed polypeptide antigen, representing the carboxy-terminal third of the external membrane glycoprotein gene fused with the amino-terminal half of the transmembrane glycoprotein gene. Over a 3-month period, 2707 consecutive serum samples referred for confirmatory testing for human T-lymphotrophic virus type III (HTLV-III) antibodies were evaluated by both Western blot and CBre3-EIA. On a single determination for each sample, the CBre3-EIA was found to have an estimated sensitivity (99.9%) and specificity (99.1%) similar or superior to the more cumbersome Western blot method. This study shows that all HIV-seropositive subjects have antibodies to the virus envelope protein; no other virus antigens are required for construction of highly sensitive immunoassays.

Temporal expression of HIV-1 envelope proteins in baculovirus-infected insect cells: Implications for glycosylation and CD4 binding

Three different human immunodeficiency virus type I (HIV-1) envelope derived recombinant proteins and the full length human CD4 polypeptide were expressed in Spodoptera frugiperda (Sf9) cells. DNA constructs encoding CD4, gp120, gp160, and gp160 delta (full length gp160 minus the transmembrane and cytoplasmic region of gp41) were cloned into the baculovirus expression vector pVL941 or a derivative and used to generate recombinant viruses in a cotransfection with DNA from Autographa californica nuclear polyhedrosis virus (AcMNPV). Western blotting of cell extracts of the recombinant HIV-1 proteins showed that for each construct two major bands specifically reacted with anti-HIV-1 envelope antiserum. These bands corresponded to glycosylated and nonglycosylated versions of the HIV proteins as determined by 3H-mannose labeling and tunicamycin treatment of infected cells. A time course of HIV envelope expression revealed that at early times post-infection (24 hours) the proteins were fully glycosylated and soluble in nonionic detergents. However, at later times postinfection (48 hours), expression levels of recombinant protein reached a maximum but most of the increase was due to a rise in the level of the nonglycosylated species, which was largely insoluble in nonionic detergents. Thus, it appears that Sf9 cells cannot process large amounts of glycosylated recombinant proteins efficiently. As a measure of biological activity, the CD4 binding ability of both glycosylated and nonglycosylated recombinant HIV envelope proteins was tested in a coimmunoprecipitation assay. The results showed that CD4 and the glycosylated versions of recombinant gp120 or gp160 delta specifically associated with one another in this analysis. Nonglycosylated gp120 or gp160 delta proteins from tunicamycin-treated cultures did immunoprecipitate with anti-HIV-1 antiserum but did not interact with CD4. We conclude that production of native HIV envelope proteins, as measured by addition of carbohydrate side chains and ability to bind CD4, peaks early after infection in baculovirus-infected insect cells.

Safety, efficacy, and immunogenicity of a recombinant Osp subunit canine Lyme disease vaccine

A subunit canine Lyme disease vaccine formulated with recombinant lipidated Osp A and OspB and saponin QS21 was assessed for safety, protective efficacy, and immunogenicity. Ten normal beagles were subcutaneously vaccinated twice at age 12 and 16 weeks, respectively. Three months after the second vaccination, the vaccinates and another 10 nonvaccinated control beagles were challenged by feeding ticks on each dog for 5 days using eight field-collected adult female and six adult male Ixodes scapularis infected with Lyme disease spirochetes per dog. Adverse reactions associated with the vaccinations were limited to injection site swellings which occurred within the first 48 h and resolved within a week. The local reaction was independent of vaccination times and tick challenge. On the basis of typical clinical signs, xenodiagnosis, and diagnostic immunoblotting, all 10 controls were infected; five developed lameness and three of them experienced at least two to three episodes of limping during a 10-month monitoring period. In contrast, eight of ten vaccinates were protected and two infected vaccinates, as judged by xenodiagnosis, were asymptomatic. None of the protected vaccinates developed antibodies to diagnostic spirochetal antigens other than OspA and OspB. In contrast, most controls produced antibodies to borrelial antigens, but not to OspA and OspB. Antibody production in vaccinates receiving a third vaccination 10 months postchallenge was greatly boosted; the geometric mean antibody titer was significantly higher (P < 0.0001) than that tested prechallenge. Thus, the subunit canine Lyme disease vaccine was safe and protective and elicited immunological memory. Vaccinated dogs were serologically distinguishable from those naturally exposed.

Synthesis and processing of simian virus 40-specific RNA in adenovirus-infected, simian virus 40-transformed human cells.

Human simian virus 80 (SV80) cells transformed by simian virus 40 (SV40) synthesize substantial quantities of the SV40 large T-antigen (Henderson & Livingston, 1974; Tjian, 1978) and cytoplasmic, poly(A)-containing RNA species that exhibit spliced structures characteristic of the SV40, early messenger RNA species that encode both large and small T-antigens (Flint & Beltz, 1979). When SV80 cells were infected with type C adenovirus, both the synthesis of SV40 large T-antigen and the appearance in the cytoplasm of newly synthesized, SV40-specific RNA sequences were inhibited during the late phase of infection. The results of hybridization to SV40 DNA of SV80 nuclear RNA, prepared from mock- or adenovirus-infected cells after labeling for short periods in vivo or in vitro, indicated that transcription of integrated SV40 was, by contrast, not disrupted during the late phase of adenovirus infection. Poly(A)-containing, nuclear RNA species that hybridized to SV40 DNA sequences and exhibited the sizes of spliced, large and small T-antigen mRNA species were also synthesized in infected cells at a time when the corresponding mRNA sequences did not leave the nucleus. These results suggest that the failure of non-adenoviral mRNA sequences to enter the cytoplasm of adenovirus-infected cells does not reflect inhibition of either their transcription or the normal enzymatic processing reactions to which pre-mRNA species are subject. Several lines of evidence do, however, establish that nuclear, SV40-specific RNA sequences are less stable in adenovirus-infected compared to mock-infected SV80 cells.

Decontamination of Bacillus anthracis Spores: Evaluation of Various Disinfectants.

The present study compares the efficacy of various disinfectants against Bacillus anthracis spores. While Bleach Rite® and 10% bleach reduce spore numbers by 90% within 10 minutes, a long contact time is required for complete disinfection. By contrast, although SporGon® did not initially reduce the number of spores as quickly as Bleach Rite or 10% bleach, shorter contact times were required for complete eradication of viable spores.