Correlation of dihydrofolate reductase elevation with gene amplification in a homogeneously staining chromosomal region in L5178Y cells.

A methotrexate (MTX)-resistant murine lymphoblastoid cell line has been obtained by serial passage in increasing concentrations of MTX which is greater than 100,000-fold resistant to MTX (L5178YR) and has dihydrofolate reductase (DHFR) levels 300-fold higher than the parental line. The L5178YR cell line synthesizes approximately 10-11% of its total soluble cell protein as DHFR regardless of growth phase, as measured by direct immunoprecipitation with a monospecific antiserum. Molecular hybridization of a purified [3H]DNA probe complimentary to DHFR specific mRNA with cellular DNA and RNA indicates that DHFR coding sequences are elevated several hundred fold in both nucleic acid species in the mutant cell line. Giemsa-banding studies of the diploid mutant line indicate the presence of a large homogeneously staining region on chromosome No. 2. In situ molecular hybridization studies indicate that the DHFR genes are localized in this homogeneously staining region. The homogeneously staining region probably consists of tandom repeats of a basic segment approximately 800 kilo base pairs long.

Fusion-competent vaccines: broad neutralization of primary isolates of HIV.

Current recombinant human immunodeficiency virus (HIV) gp120 protein vaccine candidates are unable to elicit antibodies capable of neutralizing infectivity of primary isolates from patients. Here, "fusion-competent" HIV vaccine immunogens were generated that capture the transient envelope-CD4-coreceptor structures that arise during HIV binding and fusion. In a transgenic mouse immunization model, these formaldehyde-fixed whole-cell vaccines elicited antibodies capable of neutralizing infectivity of 23 of 24 primary HIV isolates from diverse geographic locations and genetic clades A to E. Development of these fusion-dependent immunogens may lead to a broadly effective HIV vaccine.

Molecular cloning and characterization of the glucoamylase gene of Aspergillus awamori.

The filamentous ascomycete Aspergillus awamori secretes large amounts of glucoamylase upon growth in medium containing starch, glucose, or a variety of hexose sugars and sugar polymers. We examined the mechanism of this carbon source-dependent regulation of glucoamylase accumulation and found a several hundredfold increase in glucoamylase mRNA in cells grown on an inducing substrate, starch, relative to cells grown on a noninducing substrate, xylose. We postulate that induction of glucoamylase synthesis is regulated transcriptionally. Comparing total mRNA from cells grown on starch and xylose, we were able to identify an inducible 2.3-kilobase mRNA-encoding glucoamylase. The glucoamylase mRNA was purified and used to identify a molecularly cloned 3.4-kilobase EcoRI fragment containing the A. awamori glucoamylase gene. Comparison of the nucleotide sequence of the 3.4-kilobase EcoRI fragment with that of the glucoamylase I mRNA (as determined from molecularly cloned cDNA) revealed the existence of four intervening sequences within the glucoamylase gene. The 5' end of the glucoamylase mRNA was mapped to several locations within a region -52 to -73 nucleotides from the translational start. Sequence and structural features of the glucoamylase gene of the filamentous ascomycete A. awamori were examined and compared with those reported in genes of other eucaryotes.

Turning a corner on HIV neutralization?

HIV vaccine development has been hampered by the inability of conventional immunogens to elicit antibodies capable of neutralizing primary isolates of the virus. Recent studies using 'fusion-competent' immunogens that capture transitional intermediate structures of the functioning envelope protein suggest that this goal may now be achievable.

Feline leukemia virus envelope protein expression encoded by a recombinant vaccinia virus: apparent lack of immunogenicity in vaccinated animals.

We have constructed a recombinant vaccinia virus encoding the expression of the feline leukemia virus (FeLV) envelope gene of the Gardner-Arnstein strain of FeLV subgroup B. Human cells infected with the recombinant virus (vFeLVenv) express and process the FeLV envelope protein similarly to cells infected with authentic FeLV. The mature gp 70 protein is transported to and accumulates on the surface of vFeLVenv-infected cells. Vaccinia virus replication and FeLV gp70 accumulation was also observed in cells of feline and murine origin, albeit at levels somewhat reduced from those in human cells. Toward the goal of developing a recombinant vaccinia virus as a live vaccine for feline leukemia disease in cats, immunogenicity studies were performed in cats and mice. These experiments yielded surprising results: although animals mounted a typical virus-neutralizing antibody response to the vaccinia virus vector, we were unable to detect antibodies against FeLV gp70 in any of the vaccinated animals. A subsequent 'booster' immunization with killed FeLV was unable to elicit evidence of immunologic 'priming' by the recombinant virus. We are presently unable to explain the apparent lack of immunogenicity. These results may point to complexities involved in the development of vaccines to protect against retrovirus infection.

Development of a single-shot subunit vaccine for HIV-1.

The successful development of an AIDS vaccine will require formulations that not only invoke the desired immunological response, but also are stable and easy to administer. A single shot MN rgp120 vaccine formulation comprised of MN rgp120 encapsulated in poly (lactic-coglycolic) acid (PLGA) microspheres was developed to provide an in vivo autoboost of antigen. These formulations were designed to yield an in vivo autoboost at 1, 2, 3 or 4-6 months. In addition, PLGA microspheres containing the adjuvant, QS21, were also prepared to provide an in vivo autoboost concomitant with antigen. In guinea pigs, these formulations yielded higher anti-MN rgp120 and anti-V3 loop antibody titers than alum formulations that were administered at higher antigen doses. Different doses of encapsulated MN rgp120 provided a clear and well-defined dose response curve for both anti-MN rgp120 and anti-V3 loop antibody titers. When soluble QS21 was mixed with the encapsulated MN rgp120, the antibody titers were increased by a factor of 5 over the titers with encapsulated MN rgp120 alone. An additional fivefold increase in antibody titers was observed for guinea pigs immunized with encapsulated MN rgp120 and QS21 on the same microspheres. These results suggest that the adjuvant properties of QS21 can be increased by microencapsulation in PLGA. Furthermore, antibodies induced by these preparations neutralized the MN strain of HIV-1. The neutralization titers for sera from animals immunized with MN rgp120-PLGA and soluble QS21 were greater than the titers obtained from guinea pigs that were treated with MN rgp120 and soluble QS21 at the same dose. Overall, these studies validate the in vivo autoboost concept, reveal a method for improving the adjuvant properties of QS21, and indicate the potential of future single shot vaccine formulations.

Immunogenicity and HIV-1 virus neutralization of MN recombinant glycoprotein 120/HIV-1 QS21 vaccine in baboons .

The effect of adjuvant and immunization schedule on the immunogenicity of HIV-1 envelope glycoprotein, MN rgp120, was optimized by using baboons. The novel adjuvant QS21 elicited earlier seroconversion than alum adjuvant, and the antibody titers to MN rgp120 for animals treated with QS21 were significantly greater than the titers obtained in animals treated with alum. The use of QS21 shifted the dose-response curve, resulting in less MN rgp120 required to achieve equivalent titers to those in the alum formulations. The in vitro virus neutralizing (VN) titers from animals treated with QS21 were 3- to 10-fold higher than with alum. The data presented herein point to the superiority of QS21 as adjuvant in primates for MN rgp120.

Controlled release of interleukin-2 from biodegradable microspheres.

We have evaluated the use of biodegradable poly(DL-lactide-co-glycolide) microspheres for the controlled release of interleukin-2 (IL-2) and its modified forms: succinyl IL-2 (SIL-2) and polyethylene glycol-modified IL-2 (PEG IL-2). We show that a microsphere formulation can be prepared from PEG IL-2 using HSA as an excipient which, after an initial burst, releases 2-3% PEG IL-2 per day in a bioactive form continuously over a 20- to 30-day period.

Nucleotide sequences of the envelope genes of two isolates of feline leukemia virus subgroup B.

We determined the nucleotide sequences of the envelope genes of the Snyder-Theilen and Gardner-Arnstein isolates of feline leukemia virus subgroup B. Comparison of the deduced amino acid sequences of the envelope gene products revealed regions of sequence divergence, which we relate to structural features of the viral protein. We also examined nucleotide sequences within the long terminal repeats of these related isolates of feline leukemia virus subgroup B.

Recombinant feline herpesviruses expressing feline leukemia virus envelope and gag proteins.

We constructed recombinant feline herpesviruses (FHVs) expressing the envelope (env) and gag genes of feline leukemia virus (FeLV). Expression cassettes, utilizing the human cytomegalovirus immediate-early promoter, were inserted within the thymidine kinase gene of FHV. The FeLV env glycoprotein expressed by recombinant FHV was processed and transported to the cell surface much as in FeLV infection, with the exception that proteolytic processing to yield the mature gp70 and p15E proteins was less efficient in the context of herpesvirus infection. Glycosylation of the env protein was not affected; modification continued in the absence of efficient proteolytic processing to generate terminally glycosylated gp85 and gp70 proteins. A recombinant FHV containing the FeLV gag and protease genes expressed both gag and gag-protease precursor proteins. Functional protease was produced which mediated the proteolytic maturation of the FeLV gag proteins as in authentic FeLV infection. Use of these recombinant FHVs as live-virus vaccines may provide insight as to the role of specific retroviral proteins in protective immunity. The current use of conventional attenuated FHV vaccines speaks to the wider potential of recombinant FHVs for vaccination in cats.

Adaptation to persistent growth in the H9 cell line renders a primary isolate of human immunodeficiency virus type 1 sensitive to neutralization by vaccine sera.

Seven diverse primary isolates of human immunodeficiency virus type 1 (HIV-1) were examined and found to be refractory to neutralization by antisera to recombinant gp120 (rgp120) protein from HIV-1 MN. This stands in marked contrast to the sensitivity exhibited by certain laboratory-adapted viruses. To understand the difference between primary and laboratory-adapted viruses, we adapted the primary virus ACH 168.10 to growth in the FDA/H9 cell line. ACH 168.10 was chosen because the V3 region of gp120 closely matches that of MN. After 4 weeks, infection became evident. The virus (168A) replicated in FDA/H9 cells with extensive cytopathic effect but was unchanged in sensitivity to antibody-mediated neutralization. Thus, growth in cell lines is not sufficient to render primary virus sensitive to neutralization. The 168A virus was, however, partially sensitive to CD4 immunoadhesin (CD4-Ig). Adaptation was continued to produce a persistently infected FDA/H9 culture that displayed minimal cytopathic effect. The virus (168C) was now sensitive to neutralization by MN rgp120 vaccine sera and by MN-specific monoclonal antibodies and showed increased sensitivity to HIVIG and CD4-Ig. 168C encoded three amino acid changes in gp120, including one within the V3 loop (I-166-->R, I-282-->N, G-318-->R). MN-specific monoclonal antibodies bound equally to the surface of cells infected with either neutralization-resistant or -sensitive virus. The coincidence of changes in neutralization sensitivity with changes in cell tropism and cytopathic effect suggests a common underlying mechanism(s) acting through the whole of the envelope protein complex.

Method to map antigenic determinants recognized by monoclonal antibodies: localization of a determinant of virus neutralization on the feline leukemia virus envelope protein gp70.

A method is presented whereby antigenic determinants recognized by specific monoclonal antibodies can be mapped to specific sites on a protein sequence with high resolution. Short DNase I-generated DNA fragments encoding portions of the protein of interest are molecularly cloned into the EcoRI site of the beta-galactosidase gene of phage lambda Charon 16 so as to obtain expression of random protein fragments as fusion proteins. The monoclonal antibody is used to screen the phage library to isolate phage expressing the specific antigenic determinant. DNA of immunoreactive phage can be analyzed rapidly and subcloned to allow DNA sequence determination. The method is generally applicable and permits antigenic determinants of functionally interesting monoclonal antibodies to be mapped and related to specific protein sequences. We have used this procedure to determine the region of the feline leukemia virus envelope protein gp70 recognized by a virus-neutralizing monoclonal antibody, cl.25. Antibody binding was mapped to a 14-amino acid region in the amino-terminal half of gp70. This region may be directly involved in an essential function of the gp70 protein, perhaps in gp70-mediated host recognition functions. Synthetic peptides derived from this region may provide useful vaccine antigens for the prevention of feline leukemia virus-associated disease in cats.

Size heterogeneity in the 3' end of dihydrofolate reductase messenger RNAs in mouse cells.

We have examined in detail the RNA coding for dihydrofolate reductase (DHFR) in methotrexate-resistant mouse cells. We find four distinct DHFR messengers, ranging in size from 750 to 1600 nucleotides. All four are polyadenylated and polysomal and can be translated in vitro to produce a 21,000 dalton protein co-migrating with purified dihydrofolate reductase on SDS polyacrylamide gels. The major difference in these RNAs is the length of 3' untranslated regions, varying from about 80 nucleotides in the smallest mRNA to about 930 nucleotides in the largest. The RNAs are also present in methotrexate-sensitive murine cells and mouse liver. Multiple DHFR RNAs are found in the poly(A)+ RNA of methotrexate-resistant Chinese hamster ovary cells but are of different molecular weights than the mouse messengers. We discuss these results in terms of the function of 3' untranslated regions of eucaryotic mRNAs and the possible origin and significance of multiple messenger RNAs for a single protein.

Continued utilization of CCR5 coreceptor by a newly derived T-cell line-adapted isolate of human immunodeficiency virus type 1.

The differential use of CC chemokine receptor 5 (CCR5) and CXC chemokine receptor 4 (CXCR4) may be intimately involved in the transmission and progression of human immunodeficiency virus infection. Changes in coreceptor utilization have also been noted upon adaptation of primary isolates (PI) to growth in established T-cell lines. All of the T-cell line-adapted (TCLA) viruses studied to date utilize CXCR4 but not CCR5. This observation had been suggested as an explanation for the sensitivity of TCLA, but not PI, viruses to neutralization by recombinant gp120 antisera and V3-directed monoclonal antibodies, but recent studies have shown coreceptor utilization to be independent of neutralization sensitivity. Here we describe a newly isolated TCLA virus that is sensitive to neutralization but continues to utilize both CXCR4 and CCR5 for infection. This finding further divorces coreceptor specificity from neutralization sensitivity and from certain changes in cell tropism. That the TCLA virus can continue to utilize CCR5 despite the changes that occur upon adaptation and in the apparent absence of CCR5 expression in the FDA/H9 T-cell line suggests that the interaction between envelope protein and coreceptor may be mediated by multiple weak interactions along a diffuse surface.

Structural and functional analysis of the HIV gp41 core containing an Ile573 to Thr substitution: implications for membrane fusion.

The envelope glycoprotein of HIV-1 consists of the surface subunit gp120 and the transmembrane subunit gp41. Binding of gp120 to target cell receptors induces a conformational change in gp41, which then mediates the fusion of viral and cellular membranes. A buried isoleucine (Ile573) in a central trimeric coiled coil within the fusion-active gp41 ectodomain core is thought to favor this conformational activation. The role of Ile573 in determining the structure and function of the gp120-gp41 complex was investigated by mutating this residue to threonine, a nonconservative substitution in HIV-1 that occurs naturally in SIV. While the introduction of Thr573 markedly destabilized the gp41 core, the three-dimensional structure of the mutant trimer of hairpins was very similar to that of the wild-type molecule. A new hydrogen-bonding interaction between the buried Thr573 and Thr569 residues appears to allow formation of the trimer-of-hairpins structure at physiological temperature. The mutant envelope glycoprotein expressed in 293T cells and incorporated within pseudotyped virions displayed only a moderate reduction in syncytium-inducing capacity and virus infectivity, respectively. Our results demonstrate that the proper folding of the gp41 core underlies the membrane fusion properties of the gp120-gp41 complex. An understanding of the gp41 activation process may suggest novel strategies for vaccine and antiviral drug development.

Amplified dihydrofolate reductase genes are localized to a homogeneously staining region of a single chromosome in a methotrexate-resistant Chinese hamster ovary cell line.

Methotrexate-resistant Chinese hamster ovary cells selected for high resistance by progressive increments of methotrexate in the culture medium have levels of dihydrofolate reductase (tetrahydrofolate dehydrogenase, 7,8-dihydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) 200 times that of sensitive cells and a corresponding increase in the number of copies of the dihydrofolate reductase gene. The resistant cells contain an expanded region on the second chromosome (homogeneously staining region) that is not present in sensitive cells. In situ hybridization of DNA complementary to dihydrofolate reductase mRNA shows that the dihydrofolate reductase genes are specifically localized to the homogeneously staining region of this chromosome in the resistant cells.

Interleukin 2 acts as an adjuvant to increase the potency of inactivated rabies virus vaccine.

Interleukin 2 (IL-2) occupies a central position in the cascade of events involved in the immune response. We were interested in determining whether IL-2 could function as an adjuvant to vaccination, to increase the immune response to vaccine immunogens. Using the National Institutes of Health test for rabies vaccine potency, we found that daily systemic administration of IL-2 in conjunction with inactivated rabies virus can increase the potency of vaccination in outbred mice at least 25-fold, as measured by survival following challenge with virulent rabies virus. Enhanced protection is not correlated with an increase in virus-neutralizing antibody titers, and we suggest that IL-2 acts to increase the cellular immune response to vaccination.

Structure and genomic organization of the mouse dihydrofolate reductase gene.

The genomic organization of the mouse dihydrofolate reductase gene has been determined by hybridization of specific cDNA sequences to restriction endonuclease-generated fragments of DNA from methotrexate-resistant S-180 cells. The dihydrofolate reductase gene contains a minimum of five intervening sequences (one in the 5' untranslated region and four in the protein-coding region) and spans a minimum of 42 kilobase pairs on the genome. Genomic sequences at the junction of the intervening sequence and mRNA-coding sequence and at the polyadenylation site have been determined. A similar organization is found in independently isolated methotrexate-resistant cell lines, in the parental sensitive cell line and in several inbred mouse strains, indicating that this organization represents that of the natural gene.