Dani P. Bolognesi

Potent suppression of HIV-1 replication in humans by T-20, a peptide inhibitor of gp41-mediated virus entry.

T-20, a synthetic peptide corresponding to a region of the transmembrane subunit of the HIV-1 envelope protein, blocks cell fusion and viral entry at concentrations of less than 2 ng/ml in vitro. We administered intravenous T-20 (monotherapy) for 14 days to sixteen HIV-infected adults in four dose groups (3, 10, 30 and 100 mg twice daily). There were significant, dose-related declines in plasma HIV RNA in all subjects who received higher dose levels. All four subjects receiving 100 mg twice daily had a decline in plasma HIV RNA to less than 500 copies/ml, by bDNA assay. A sensitive RT–PCR assay (detection threshold 40 copies/ml) demonstrated that, although undetectable levels were not achieved in the 14-day dosing period, there was a 1.96 log10 median decline in plasma HIV RNA in these subjects. This study provides proof-of-concept that viral entry can be successfully blocked in vivo. Short-term administration of T-20 seems safe and provides potent inhibition of HIV replication comparable to anti-retroviral regimens approved at present.

Neutralizing Antibody Responses to Human Immunodeficiency Virus Type 1 in Primary Infection and Long-Term-Nonprogressive Infection

The role of neutralizing antibodies in human immunodeficiency virus type 1 (HIV-1) infection is poorly understood and was assessed by evaluating responses at different stages of infection. Undiluted sera from long-term nonprogressors (LTNP) had broad neutralizing antibodies against heterologous primary isolates and were more likely to neutralize the contemporaneous autologous isolate than were sera from short-term nonprogressors and progressors. In primary infection, envelope-specific IgG was detected before the initial decline in plasma viremia, but neutralizing antibodies developed more slowly. Here, neutralizing antibodies against strains SF-2 and MN were sometimes the first to be detected, but titers were low for at least 17 weeks from onset of symptoms. Neutralizing antibodies against the early autologous isolate were detected for 4 patients by 5-40 weeks but were undetectable in 2 additional patients for 27-45 weeks. The results indicate that neutralizing antibody responses are slow to develop during primary infection and are uniquely broad in LTNP.

Broadly neutralizing antibodies elicited by the hypervariable neutralizing determinant of HIV-1

The principal neutralizing determinant (PND) of human immunodeficiency virus (HIV)-1 resides within the V3 loop of the envelope protein. Antibodies elicited by peptides of this region were able to neutralize diverse isolates. Serum from one of three animals immunized with the human T cell lymphoma virus (HTLV)-IIIMN PND peptide, RP142, neutralized MN and the sequence-divergent HTLV-IIIB isolate. Serum from one of three animals immunized with a 13-amino acid IIIB PND peptide (RP337) also neutralized both of these isolates. Characterization of these sera revealed that the cross-neutralizing antibodies bound the amino acid sequence GlyProGlyArgAlaPhe (GPGRAF) that is present in both isolates. This sequence is frequently found in the PNDs analyzed in randomly selected HIV-1 isolates. Sera from two rabbits immunized with a peptide containing only the GPGRAF residues neutralized divergent isolates, including IIIB and MN.

Enfuvirtide: the first therapy to inhibit the entry of HIV-1 into host CD4 lymphocytes

Highly active antiretroviral therapy (HAART) based on combinations of drugs that target key enzymes in the life-cycle of human immunodeficiency virus (HIV) has considerably reduced morbidity and mortality from HIV infection since its introduction in the mid-1990s. However, the growing problem of the emergence of HIV strains that are resistant not only to individual drugs, but to whole drug classes, means that agents with new mechanisms of action are needed. Here, we describe the discovery and development of enfuvirtide (Fuzeon), the first drug to inhibit the entry of HIV-1 into host cells.

EVIDENCE FOR HUMAN INFECTION WITH AN HTLV III/LAV-LIKE VIRUS IN CENTRAL AFRICA, 1959

An individual serum stored since 1959 from Central Africa has been demonstrated reactive against HTLV-III by ELISA Western blotting and by immunoprecipitation against HTLV-III as well as STLV-III. This serum was from a set of 1213 plasmas from various parts of Africa 818 dating from 1959. Because of the known high false positive rates on Abbott EIA in long-term frozen sera those found to be above the cutoff using the formula (mean of 3 negative serum controls plus 10% of the mean of 2 positive serum controls) x 3 were retested by immunofluorescence microscopy and Western blot enzymatic immunoassay. 1 serum taken from Central Africa in 1959 was positive by both tests. This plasma had an OD more than 7 x the cutoff value. It reacted strongly in immunofluorescence with infected H9 cells. Reactivity was detected by Western Blot against all major HTLV-III viral proteins and polypeptide 121. By immunoprecipitation it reacted with all major gag and env encoded proteins of HTLV-III and the gag encoded proteins of STLV-III-AGM. These results suggest that HTLV-III prevalence was low in 1959 but that at least 1 individual was exposed to a similar virus over 25 years ago in Central Africa.

Polypeptides of avian RNA tumor viruses: I. Isolation and physical and chemical analysis

Abstract Extraction of avian myeloblastosis virus with phenol and SDS and electrophoresis of the proteins on polyacrylamide gels resulted in a variety of components. Four major bands were detected by protein specific staining and their molecular weights were estimated to be 13,000, 15,000, 23,000, and 28,000. These components were shown to be present in virus from both subgroup A and B. One of these (15,000 mol. wt.) demonstrated exceptional staining properties with amido black but incorporated radioactive amino acids poorly. Another component (23,000 mol. wt.) showed a tendency to aggregate which could be eliminated by use of dithiothreitol. In addition to these major components a series of smaller bands of higher molecular weight were detected. The most distinct of these (115,000 mol. wt.) seemed to be associated with material which could be stained for polysaccharide and was strongly labeled with glucosamine- 14 C. A similar component was also found in preparations known to contain the type-specific antigen of the virus. The various components could be recovered following preparative electrophoresis enabling immunological analysis to be performed (Bauer and Bolognesi, this series, Part II, see following article).

Localization of RNA tumor virus polypeptides: I. Isolation of further virus substructures

Abstract The internal components of avian myeloblastosis virus (AMV) and Friend murine leukemia virus (FLV) were isolated and characterized. AMV cores contained three of the five major virus polypeptides having molecular weights of 27,000, 15,000, and 12,000. FLV cores consisted of three of the four major components, and these have molecular weights of 31,000, 15,000, and 10,000. For both types of cores the 15,000 dalton component was present in least amounts. The internal ribonucleoprotein was isolated from each of the cores and shown to consist of the most basic protein of the respective agents. The properties of the proteins and their location in the virions indicated a strong similarity between the avian and mammalian agents.

Changes in bioactive lipids, alkylacylglycerol and ceramide, occur in HIV-infected cells

The mass levels of bioactive lipids known to modulate signal transduction or to possess other biological activities were measured in HIV-infected CEM cells. The levels of diacylglycerol, an activator of protein kinase C, as well as of alkylacylglycerol were elevated. A more drastic increase was observed in the ceramide levels after HIV-infection, whereas sphingosine levels were hardly influenced. Interestingly, the magnitude of the changes was related to the infection time, being higher at 8 days after infection then at 4 days. The possible role of these lipids in the cytopathic effects of HIV-infection is discussed. In addition, an improved methodology to quantitate simultaneously diacylglycerol and alkylacylglycerol in crude lipid extracts, based upon their phosphorylation by E. coli diacylglycerol kinase, is presented.

Polypeptides of avian RNA tumor viruses. IV. Components of the viral envelope

Abstract By treatment of avian myeloblastosis virus (AMV) with NP 40, it was possible to quantitatively isolate a rosettelike structure whose subunits are related to the surface projections of the virus. Detailed analysis of this material resulted in the following findings: (i) The rosettes represented about 7% of the protein and 60% of the carbohydrate content of the virus. (ii) SDS-polyacrylamide gel electrophoresis indicated the presence of two glycoproteins having molecular weights of 37,000 (GI) and 115,000 (GII). (iii) GII could be isolated without treatment with SDS and structurally resembled the knob portion of the rosettes. (iv) Both GI and GII specifically absorbed neutralizing sera and precipitated with these in immunodiffusion analysis. (v) In addition to type-specific antigenicity, two distinct antigenic determinants were demonstrated in the rosettes utilizing nonneutralizing rabbit sera. One of these resided in GI and the other in GII. These antigens were shown to be distinct from the major complement-fixing antigens of the virus. These results confirm the belief that the surface projections of avian RNA tumor viruses represent the type-specific antigens.

Polypeptides of mammalian oncornaviruses: I. Isolation and serological analysis of polypeptides from murine and feline C-type viruses

Abstract Agents representative of murine and feline C-type viruses contain four major polypeptides of molecular weights 10,000 (P1), 12,000 (P2), 15,000 (P3), and 31,000 (P4). Each was shown to contain distinct antigenic determinants. P1 of Friend leukemia virus (FLV) demonstrates species-specific reactivity different from that present in FLV P4. It also seems to be associated with a host cell component. FLV P2 revealed type or subgroup specificity and was shown to be associated with carbohydrate. FLV P3 was not investigated in as much detail. Feline leukemia virus P4, which is known to contain both species and interspecies determinants, revealed also a type-or subgroup-specific reactivity. Finally, an antiserum prepared against murine P4 was shown to contain antibodies directed against distinguishable interspecies determinants.