ISSN:0889-2229    

DOI:10.1089/aid.1991.7.261

年代:1991

数据来源: MAL

摘要:

In order to examine thein vivoprevalence of AZT resistance mutations in AIDS patients after long-term therapy we amplified, by polymerase chain reaction (PCR), a 654 bppolgene fragment from peripheral blood mononuclear cell DNA samples from a patient before, and 19 months after, the start of AZT therapy. PCR products from each sample were cloned and 9 clones from each sample were sequenced. Seven of 9 clones from the post-AZT sample, but none from the pre-AZT sample, contained an amino acid substitution (Thr215to Tyr) requiring two nucleotide changes within the same codon (ACC to TAC). This change had previously been shown by Larder and Kemp (Science, 246:1155-1158,1989) to correlate with partial AZT resistance of virusisolates. In colony hybridizations using synthetic oligonucleotides corresponding to the mutant and wild-type sequences, 22 of 22 clones from the pre-AZT sample hybridized only to the wild-type probe while 21 of 26 clones from the post-AZT sample hybridized only to the mutant. Clinically, this patient remains well, indicating that while Tyr215may be the first amino acid substitution leading to resistance, it alone does not appear to have significantly influenced the clinical status of this patient.

ISSN:0889-2229    

DOI:10.1089/aid.1991.7.265

年代:1991

数据来源: MAL

摘要:

The aim of this study was to determine if protection against an infectious human immunodeficiency virus type 2 (HIV-2) challenge could be obtained in cynomolgus macaques by active immunization using whole killed virus vaccine. Four monkeys were immunized with killed HIV-2SBL-6669, two of them with five intramuscular (im) injections of viral preparation containing 100 or 300 μg protein emulsified in incomplete Freund's adjuvant (IFA) and the two remaining received four im injections of 25-50 μg viral protein in iscoms. Each of the four vaccinated cynomolgus monkeys, along with four unvaccinated controls, were challenged intravenously two weeks after the last booster with approximately 100 animal infectious doses (ID50) of live HIV-2SBL-6669. All four immunized monkeys developed antibodies to HIV-2 envelope and core proteins before challenge exposure to HIV-2, but only the two animals vaccinated with virus in IFA developed detectable neutralizing antibodies. The two monkeys immunized with killed virus in IFA have shown no evidence of infection nine months after challenge with live virus. When blood and lymph node cells from these animals were transfused into naive cynomolgus monkeys, the recipients remained free of infection. In contrast, virus was recovered repeatedly in all nonimmunized animals and in the two animals immunized with iscom-associated viral antigens, which had a low content of envelope gp125 antigen. The demonstration of vaccine-induced protection against HIV-2 in a nonhuman primate raises hope for effective immunization against HIV infections in humans as wel

ISSN:0889-2229    

DOI:10.1089/aid.1991.7.271

年代:1991

数据来源: MAL

摘要:

The occurrence of dominant linear antigenic sites in the envelope glycoproteins of human immunodeficiency virus type 2 (HIV-2) was evaluated. Twenty-five peptides representing different regions of HIV-2, strain SBL-6669, were synthesized. For comparison the corresponding peptides of HIV-1, strain BRU, were also prepared. The peptides were tested in enzyme-linked immunosorbent assay (ELISA) with human sera from individuals with proven HIV-1 or HIV-2 infection and simian sera from animals infected with HIV-2 or simian immunodeficiency virus of sooty mangabay monkey origin (SIVsm). Four major antigenic regions were identified. Peptides representing parts or the whole V3 (neutralizing loop) region and an additional stretch of amino acids located at the carboxy terminal of this region showed considerable reactivity. This reaction was predominantly type specific, but some heterotypic reactivity was also seen. Peptides representing the carboxy terminal 21 amino acids of the V3 region of the type-related viruses HIV-2 and SIVsmallowed selective identification of strain-specific antibodies. A second major antigenic region was found close to the carboxy terminal end of the large glycoproteins. This region was cross-reactive between the two types. The two additional dominating antigenic regions were located in the amino terminal region of the transmembrane glycoprotein. One region has previously been shown to be a uniquely antigenic type-specific site. The other region was also type-specific, but was identified only in HIV-2, amino acids Glu634-Lys649. Excellent facilities are available for the design of not only type-unique site-specific serological tests but potentially also type-cross-reactive and strain-specific assays.

ISSN:0889-2229    

DOI:10.1089/aid.1991.7.279

年代:1991

数据来源: MAL

摘要:

Deletions were constructed within a functional human immunodeficiency virus type 1 (HIV-1) proviral clone in order to assess the role of the envelope protein in virus particle formation. A graded exonuclease deletion technique was used to produce 12 clones with deletions of 175-308 nucleotides in the first conserved domain of envelope. This included 9 clones with frameshift deletions and 3 clones with in-frame deletions. Isogenic pairs ofenvdeletion clones were produced with or without an additional deletion in thevifandvprgenes. Upon transfection, all clones produced virus particles, as determined by p24 antigen, reverse transcriptase, and sucrose gradient assays with conditioned media. Virus particles produced from clones with deletions inenvorvifandvpr, or both regions, banded on sucrose gradients with a mobility similar to that of virus produced by the parental clone. The p24gagcapsid protein in the particles was resistant to trypsin, but the particles were disrupted by treatment with Triton X-100, suggesting the presence of a surrounding lipid bilayer. Furthermore, electron microscopic studies revealed both mature and immature virus particles derived from COS cells transfected with theenvdeletion clones. Cocultivation experiments with lymphoid cells and cells transfected with each of theenvdeletion clones demonstrated that the virus particles were noninfectious.

ISSN:0889-2229    

DOI:10.1089/aid.1991.7.287

年代:1991

数据来源: MAL

摘要:

In characterizing a group of independent human immunodeficiency virus (HIV-1) isolates, we noted that certain isolates had anomolously low levels of virion-associated reverse transcriptase activity. In an attempt to understand the basis of this phenomenon, we examined in detail one such isolate, HIV-1G. We found correctly processed forms of the viral reverse transcriptase in virions as well as processed forms of other viral proteins, suggesting that viral proteins are both expressed and properly processed. We have detected a nuclease activity associated with the outer face of the HIV-1Genvelope. This nuclease degrades the DNA product generated during the reverse transcription assay. The nuclease activity is more sensitive to mild protein denaturation than is the viral reverse transcriptase, and it is stimulated by the presence of Ca2+. The amount of virion-associated nuclease activity relative to reverse transcriptase activity varies between virus isolates and can vary also for one isolate during virus spread through a culture. The origin of the nuclease activity is unknown but is presumed to be cellular. The variability in amount of nuclease activity may reflect variability in the interaction of the virus with different cellular components during maturation.

ISSN:0889-2229    

DOI:10.1089/aid.1991.7.295

年代:1991

数据来源: MAL

摘要:

Subcellular localization of input human immunodeficiency virus type 1 (HIV-1)gagproteins was determined in infected H9 and Jurkattatcells. Infected cells were fractionated at intervals, and the proteins in cell fraction were identified by immunoblotting using pooled sera from acquired immunodeficiency syndrome (AIDS) patients and monoclonal antibodies. Cycloheximide was added at 0 time to prove that the proteins detected were not nascent ones.Gagproteins p55, p41, p39 (in the most essential relative concentrations), and p17 were found in the cell nuclei for at least 4 h after infection. However, p24 was not found in the cell nuclei and was accumulated in the nuclear washing buffer. The data presented confirm the presence of karyotypic signal at the N terminus of p55gagprecursor. The potential role of nuclear localization ofgagprecursor is discussed.

ISSN:0889-2229    

DOI:10.1089/aid.1991.7.303

年代:1991

数据来源: MAL

摘要:

Purified recombinant reverse transcriptase (RT) from human immunodeficiency virus type 1 (HIV-1) was used to raise 21 monoclonal antibodies with anti-RT specificities. The antibodies were characterized using Western blotting against native virus and recognized either the p66 or p66, p51 components of RT. Further immunoblotting using either cyanogen bromide fragmented RT or truncated mutants of RT along with cross-competition studies enabled the location of various immunogenic regions of RT to be identified. Three antibodies recognized a linear epitope in the N-terminal region (amino acids 128-176). Also, a neutralizing RT antibody recognized a conformational epitope in this region. Three monoclonals had epitopes mapped to linear sequences in the RNase H region at the C-terminus of the RT. Another neutralizing antibody, also requiring folding of the RT protein had its epitope more centrally located (231-353). Of the remaining 13 monoclonals, 7 were roughly located in the C -terminal region and required folding of the protein for epitope recognition and only three of the remaining six could be mapped to conformational epitopes in N-terminal and central regions of the RT. None of the antibodies tested recognized HIV-2 RT products p68 and p55 in Western blot.

ISSN:0889-2229    

DOI:10.1089/aid.1991.7.307

年代:1991

数据来源: MAL

摘要:

Monoclonal antibodies have been generated against a synthetic peptide of thenefprotein of human immunodeficiency virus type 1 (HIV-1) in order to further characterize the biochemical and functional nature of this protein and its role in the control of HIV-1 transcriptional regulation. Earlier studies indicatednefto be a negative regulatory factor for viral transcription, whereas more recent studies report evidence against this original hypothesis.Nefis a protein of 206 amino acids of approximately 27 kD in most HIV-1 isolates; however, in some other isolates a truncated form of 124 amino acids has been described. A peptide sequence of six amino acids, corresponding to a region of thenefprotein exhibiting high-sequence homology to thymosin a1protein, has been synthesized by Merrifield solid-phase methodology. This peptide is coded by a sequence located upstream to the stop codon described in some HIV-1 isolates and then is maintained in both complete and truncated forms of thenefprotein. F14.11 is anefpeptide-specific monoclonal antibody (IgG2a/k) exhibiting the ability to recognize naturalnefprotein in either radioimmunoassay, radioimmunoprecipitation assay, or immunocytochemical analysis. Since F14.11 is able to identifynefprotein in the cytoplasm of lymphocytes from HIV-infected seronegative subjects it may prove useful in monitoring the expression ofnefduring the silent HIV-1 infection.

ISSN:0889-2229    

DOI:10.1089/aid.1991.7.315

年代:1991

数据来源: MAL

摘要:

In a preliminary study, two of four rabbits infected with human T-cell leukemia virus type I (HTLV-I) demonstrated prolonged primary chancres following super infection withTreponema pallidum, the causative agent of syphilis. Two rabbits inoculated with 1 X 107HTLV-I-infected human MT-2 cells and two with infected rabbit cells from a line established in this laboratory (RLT-P), developed latent HTLV-I infection as detected by seroconversion 10 weeks after infection and by detection of HTLV-I sequences in the DNA of peripheral blood lymphocytes after amplification by polymerase chair reaction (PCR) 15 weeks after infection. The rabbits remained clinically normal and had normal blood counts. Six months after infection, the four HTLV-infected rabbits and two noninfected controls were challenged by the intradermal inoculation of 1 X 106Treponema palliduminto eight sites on the shaved back. The lesions of two of the HTLV-I-infected rabbits had a time course similar to non-HTLV-I-infected controls and were completely healed by 4 weeks. The lesions of one of the other two rabbits with progressive disease began to heal about 7 weeks afterT. pallidumchallenge. The cutaneous lesions in the other rabbit remained dark-field positive and became a confluent eschar at 8 weeks; healing only after treatment with penicillin. Four months after the primary challenge none of the six rabbits previously challenged withT. pallidumhad developed lesions after rechallenge and thus expressed chancre immunity. These results demonstrate that rabbits with latent HTLV-I infections may have defective cell-mediated immunity.

ISSN:0889-2229    

DOI:10.1089/aid.1991.7.323

年代:1991

数据来源: MAL