Human immunodeficiency virus type-1 (HIV-1) Pr55gag virus-like particles are potent activators of human monocytes

Human immunodeficiency virus type-1 (HIV-1) Pr55^Gag virus-like particles (VLP) represent an interesting HIV vaccine component since they stimulate strong humoral and cellular immune responses. We demonstrated that VLP expressed by recombinant baculoviruses activate human PBMC to release pro-inflammatory (lL-6, TNF-α), anti-inflammatory (IL-10) and Th1-polarizing (IFN-γ) cytokines as well as GM-CSF and MIP-1α in a dose-and time-dependent manner. Herein, residual baculoviruses within the VLP preparations showed no or minor effects. Monocytes could be identified as a main target for VLP to induce cytokine production. Furthermore, VLP-induced monocyte activation was shown by upregulation of molecules involved in antigen presentation (MHC II, CD80, CD86) and cell adhesion (CD54). Exposure of VLP to serum inactivates its capacity to stimulate cytokine production. In summary, these investigations establish VLP as strong activators of PBMC and monocytes therein, potently enhancing their functionality and potency to promote an efficient immune response. This capacity makes VLP an interesting component of combination vaccines.     

The T12I mutation within the SP1 region of Gag restricts packaging of spliced viral RNA into human immunodeficiency virus type 1 with mutated RNA packaging signals and mutated nucleocapsid sequence

Specific packaging of human immunodeficiency virus type 1 (HIV-1) RNA is attributable to the high affinity of nucleocapsid (NC) sequence of Gag for the cis-acting RNA packaging signals located within the 5' un-translated region (5' UTR). Interestingly, we have previously reported that the T12I mutation (named MP2) within SP1 of Gag prevented incorporation of spliced viral RNA into mutated viruses that lacked the stem-loop 1 (SL1) RNA element (also named dimerization initiation site, DIS), suggesting a role for the SP1 sequence in viral RNA packaging. In this study, we have further tested this activity of MP2 in the context of a variety of mutations that affect viral RNA incorporation. The results showed that MP2 was able to effectively restrict packaging of spliced viral RNA into viruses containing either NC mutations R10A and K11A or mutated 5' UTR sequence, such as DeltaGU3 that lacked the 112-GUCUGUUGUGUG-123 sequence of U5, D1 that was deleted of a 27 nt fragment immediately downstream of the primer binding site (PBS), Delta(306-325) that had the SL3 RNA element removed and MD2 that was missing the 328-GGAG-331 sequence. As a result, MP2 contributed increased infectivity to the related viruses. Therefore, the MP2 mutation demonstrates a distinct role in HIV-1 RNA packaging that is neither pertained to the specific viral RNA packaging signal nor to the NC sequence.     

Human immunodeficiency virus type 1 (HIV-1) and human hematopoietic progenitor cells

Summary Besides a progressive depletion of CD4+ T-lymphocytes, other peripheral blood cytopenias, (granulocytopenia, anemia and thrombocytopenia) are frequently observed in HIV-1 seropositive individuals, especially in patients with overt AIDS. Various experimental evidences suggest that HIV-1 could play a direct role in the pathogenesis of HIV-1 related peripheral blood cytopenias, affecting the survival/proliferation capacity of hematopoietic progenitors. CD34+ human hematopoietic progenitors, however, are substantially not susceptible to HIV-1 infection either in vitro and in vivo and their defects seem rather related to an alteration of bone marrow and peripheral blood microenvironments due to the presence of soluble HIV-1 specific products.     

Decay-accelerating factor (CD55) protects human immunodeficiency virus type 1 from inactivation by human complement

HIV-1 in contrast to animal retroviruses, is not lysed by human complement, but is readily inactivated by the sera from different animal species. To identify a possible species-specific protection mechanism, HIV-1 was expressed in cells of non-human origin. Recombinant HIV-1 virions that could encode the chloramphenicol acetyltransferase (CAT) protein were produced in African green monkey COS-1 cells, mink cells and, as a control, in human HEp-2 cells and were then used to infect CD4-positive target cells. Analysis of the CAT activity of the target cells' revealed that fresh HIV-1-negative human serum reduced the infectivity of HIV-1 derived from monkey and mink cells five- to tenfold, but had no effect on HIV-1 produced in human cells. In addition, human serum efficiently lysed HIV-1 produced in non-human cells in contrast to HIV-1 originating from human cells, suggesting lysis as an important mechanism of virus inactivation. Mammalian cells are protected against lysis by homologous complement by membrane-bound regulatory molecules. Two of these complement inhibitors, namely decay-accelerating factor (DAF) and, to a lesser extent, CD59 were found on the surface of HIV-1 virions by means of a virus capture assay. Antibodies against DAF, but not against other host cell molecules found on the viral surface, efficiently blocked the resistance of HIV-1 produced in human cells to human complement. These results suggest that the acquisition of DAF during the budding process from human cells protects HIV-1 in a species-specific way against the attack of human complement.     

Human immunodeficiency virus type II (HIV-2) testing: a perspective.

Testing of blood for antibodies to the human immunodeficiency virus type 2 (HIV2) has begun. A major benefit of this testing will be the elimination of the exclusion of blood donors on the basis of geographic origin. Will this test be helpful? Present anti-HIV1 testing detects between 60 to 90 percent of those samples containing the HIV2 antibody. A Food and Drug Administration (FDA) licensed enzyme immunoassay for HIV1/HIV2 antibodies became available for clinical use in March of 1992. (There is an FDA unlicensed HIV-2 Western Blot (WB) Test). The specificity and sensitivity of either the HIV2 EIA or HIV2 WB are not well defined. In a survey of 24 million U.S. donors, no antibodies to HIV2 were detected. With the incidence of HIV2 infection being so small, it is probable that the majority of positive tests will be false-positives causing needles concern for the blood donor who must be counseled. The benefits to the blood supply remain to be evaluated.     

Evaluation of the new VERSANT HIV-1 RNA 1.0 Assay (kPCR) for quantitative detection of human immunodeficiency virus type 1 RNA

BackgroundThe VERSANT HIV-1 RNA 1.0 Assay (kPCR) for quantitative detection of HIV-1 RNA has recently been introduced.ObjectivesIn this study, the performance of the VERSANT HIV-1 RNA 1.0 Assay (kPCR) was evaluated and compared to the COBAS AmpliPrep/COBAS TaqMan HIV-1 Test, version 2.0.Study designAccuracy, linearity, interassay and intra-assay variations were determined, and a total of 196 routine clinical samples including a high number of HIV-1 subtype non-B samples were investigated.ResultsWhen accuracy of the new kit was tested, all of the quantifiable results were found to be within ?0.5 log₁₀ unit of the expected panel results. Determination of linearity resulted in a quasilinear curve up to the initial concentration of 3.4 × 10⁵ copies/mL. The interassay variation ranged from 12 to 20%, and the intra-assay variation ranged from 8 to 16%. When clinical samples were tested by the VERSANT HIV-1 RNA 1.0 Assay (kPCR) and the results were compared with those obtained by the COBAS AmpliPrep/COBAS TaqMan HIV-1 test, version 2.0, the results for 95% of all samples with positive results by both tests were found to be within ±1.0 log₁₀ unit. The viral loads for all samples measured by the Siemens and Roche assays showed a high correlation (R² = 0.94); quantitative results obtained by the Siemens assay were usually found to be lower than those obtained by the Roche assay.ConclusionsThe new VERSANT HIV-1 RNA 1.0 Assay (kPCR) proved to be suitable for use in the routine diagnostic laboratory. The time to results was similar for both of the assays.     

Naturally occurring amino acid polymorphisms in human immunodeficiency virus type 1 (HIV-1) Gag p7(NC) and the C-cleavage site impact Gag-Pol processing by HIV-1 protease

Human immunodeficiency virus type 1 (HIV-1) protease activity is targeted at nine cleavage sites comprising different amino acid sequences in the viral Gag-Pol polyprotein. Amino acid polymorphisms in protease and in regions of Gag, particularly p7(NC) and the C-cleavage site between p2 and p7(NC), occur in natural variants of HIV-1 within infected patients. Studies were designed to examine the role of natural polymorphisms in protease and to identify determinants in Gag that modulate protease processing activity. Closely related Gag-Pol regions from an HIV-1-infected mother and two children were evaluated for processing in an inducible expression system, for protease activity on cleavage-site analogues, and for impact on replication by recombinant viruses. Gag-Pol regions displayed one of three processing phenotypes based on the appearance of Gag intermediates and accumulation of mature p24(CA). Gag-Pol regions that were processed rapidly to produce p24(CA) resulted in high-level replication by recombinant viruses, while slow-processing Gag-Pol variants resulted in recombinant viruses that replicated with reduced kinetics in both T cell lines and peripheral blood mononuclear cells. Direct impact by Gag sequences on processing by protease was assessed by construction of chimeric Gag-Pol regions and by site-directed mutagenesis. Optimal protease activity occurred when Gag and Pol regions were derived from the same gag-pol allele. Heterologous Gag regions generally diminished rates and extent of protease processing. Natural polymorphisms in novel positions in p7(NC) and the C-cleavage site have a dominant effect on protease processing activity. Accumulation of Gag products after processing at the C site appears to delay subsequent cleavage and production of mature p24(CA).     

Human immunodeficiency virus type 1 fitness and tropism: concept,quantification, and clinical relevance

Two distinct aspects of human immunodeficiency virus type 1 (HIV-1) biopathology with important implications for the management of treated patients have emerged during the last decade: changes in relative viral fitness, and viral tropism. First, it has been observed that HIV-1 accumulates deleterious mutations leading to drug resistance and different degrees of reduction in relative fitness during antiretroviral therapy (ART). Although the latter normally parallel a failure of ART resulting from selection of resistant mutants, the drop in viral replication capacity may be beneficial for the host. Moreover, specific antiviral compounds aimed at reducing viral fitness could be developed. Analysis of the determinants of viral fitness in highly evolving viral populations has shown that viral extinction may also be obtained by forcing highly dynamic viral populations through increased (lethal) mutagenesis that abolishes viral replication (violation of the error threshold). It could be of great interest in the near future to address this point with strategies specifically planned at the molecular level. Furthermore, diagnostic evaluation limited to the master sequence has low predictive value in rapidly evolving viral populations. These observations, together with the evidence that all of the methodologies currently used for fitness analysis have important limitations, strongly suggest that further research is warranted. This should use highly sensitive and flexible technologies to evaluate viral fitness directly in vivo or ex vivo, not only for the dominant mutants, but also for minority variants. Second, discovery of the two main co-receptors for HIV-1, CCR5 and CXCR4, has led to a better understanding of the interaction of the viral envelope with host cells and to the development of novel therapeutic agents that inhibit viral entry. In this perspective, analysis of HIV-1 tropism has acquired a major diagnostic role.     

Interaction with human immunodeficiency virus (HIV) type 2 predicts HIV type 1 genotype

In West Africa, India, and certain regions of Europe, both human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2) are known to cocirculate. To investigate the HIV-1 subtypes involved in dual HIV-1 and HIV-2 infections, we sequenced the envelope C2-V3 region from 29 dually infected female commercial sex workers from Senegal. The majority of women (23 of 29) were infected by HIV-1 subtype A. Within the HIV-1 subtype A sequences, 14 of 23 (60.8%) clustered with the West African associated A/G recombinant form (IbNG), and 9 of 23 (39.2%) formed a separate cluster distinct from the A/G IbNG. In contrast, in HIV-1 singly infected individuals, non-IbNG subtype A was found in only 13 of 98 (13.3%). Therefore, the lack of protection and/or interaction with HIV-2 was associated with a distinct HIV-1 A genotype. These results suggest differences in the biological properties of HIV-1 genotypes and their in vivo interaction with HIV-2.     

Interaction of a noncytopathic human immunodeficiency virus type 1 (HIV-1) with target cells: efficient virus entry followed by delayed expression of its RNA and protein

Recently described noncytopathic human immunodeficiency viruses type-1 (HIV-1) form a new category within the HIV-1 family due to their unique biological properties and predominant occurrence in symptom-free individuals. To study the mechanism of noncytopathic HIV-1 infection, we compared the infectivity and life cycles of two closely related HIV-1 clones with noncytopathic (N1T-E) or cytopathic (N1T-A) properties. N1T-E virus exhibited slow kinetics of infection in T cells and monocytes. Slow infection was not due to defective virus entry, because N1T-E and N1T-A exhibited equally efficient virus-cell fusion activity and nucleocapsid internalization. Kinetic studies of N1T-E genome expression revealed low levels of viral RNA, structural proteins, and Tat protein during the first 7 days after virus entry. In contrast, cells infected with the same dose of cytopathic N1T-A virus began to express high levels of genomic RNA, structural proteins, and Tat protein within 48 hr of infection; the expression peaked on Day 5, followed by complete cell lysis. No delay in N1T-E replication, as compared to N1T-A, was observed after transfection of cloned N1T-E proviral DNA. N1T-E virus had intact Tat, Rev, and fusion functions and replicated well in chronically infected cells. These results suggest that delayed processing or expression of HIV-1 genome during the early phase of the virus replicative cycle is an important determinant in noncytopathic infection.     

Relationship between human immunodeficiency virus (HIV-1) infection and chronic periodontitis

Introduction: Current studies show that, even in the era of antiretroviral therapies, HIV-1 infection is associated with more severe and frequent refractory chronic periodontitis.

Areas covered: This review, based on a systematic analysis of the literature, intends to provide an update on factors that may be involved in the pathogenesis of periodontal disease in HIV-1-infected patients, including local immunosuppression, oral microbial factors, systemic inflammation, salivary markers, and the role of gingival tissue as a possible reservoir of HIV-1.

Expert commentary: The therapeutic revolution of ART made HIV-1 infection a chronic controllable disease, reduced HIV-1 mortality rate, restored at least partially the immune response and dramatically increased life expectancy of HIV-1-infected patients. Despite all these positive aspects, chronic periodontitis assumes an important role in the HIV-1 infection status for activating systemic inflammation favoring viral replication and influencing HIV-1 status, and also acting as a possible reservoir of HIV-1. All these issues still need to be clarified and validated, but have important clinical implications that certainly will benefit the diagnosis and management of chronic periodontitis in HIV-1-infected patients, and also contributes to HIV-1 eradication.     

Human herpes virus type 6 (HHV-6) and its in vitro effect on human immunodeficiency virus (HIV).

Human herpes virus type 6 (HHV-6) was isolated from the peripheral blood lymphocytes of a patient infected with human immunodeficiency virus (HIV). Antibodies to this herpes virus were found to be widespread among adults and children in Western Australia. Co-infection studies indicated that HIV replication was inhibited by the presence of HHV-6.     

Interactions of single and combined human immunodeficiency virus type 1 (HIV-1) DNA vaccines

DNA immunization permits evaluation of possible antagonistic or synergistic effects between the encoded components. The protein expression capacity in vitro was related to the immunogenicity in vivo of plasmids encoding the HIV-1 regulatory genes tat rev, and nef. Neither Tat nor Rev expression was influenced by co-expression in vitro of all three proteins, while Nef expression was slightly inhibited. With the combination of genes, the T-cellular responses of mice against Rev and Nef were inhibited compared with those when single gene immunization was used. No interference was detected for the Tat T-cell response. Thus, co-immunization with certain genes may result in inhibition of specific immune responses.     

Human immunodeficiency virus type 1 and hepatitis B virus transcription in peripheral blood lymphocytes from co-infected subjects

Summary We have investigated restriction fragment length polymorphism (RFLP) on the PrP gene and the frequencies of RFLP patterns in 35 healthy Suffolk sheep randomly collected. According to the combinations of PrP encoding DNA fragments generated by restriction enzymesEco RI andHind III, the RFLP patterns were classified into six types and designated as types I to VI. The frequencies of these types were as follows: I, 8.6%; II, 11.4%; III, 17.6%; IV, 11.4%; V, 28.6%; and VI, 22.9%. In 10 sheep diagnosed as having natural scrapie, RFLP types, I, III, IV, and V were determined. To examine the correlation between the RFLP type and the occurrence of scrapie, the frequencies of RFLP types in sheep infected with natural scrapie were compared with those in healthy sheep. It was found that the frequency of type I in the sheep with natural scrapie was 70%, about eight times higher than that in randomly collected healthy sheep. In the 13 experimentally infected sheep that had been used for other purposes, however, no relationship between the RFLP type and onset of scrapie was found.