Highly immunogenic human immunodeficiency viruslike particles are produced by recombinant vaccinia virus-infected cells

CV-1 cells were infected with two recombinant vaccinia viruses carrying the gag gene with deletion of 231 bp from 3' terminus (strain vC5) and env gene (strain vE234L) of human immunodeficiency virus type 1 (HIV-1). Both recombinant proteins synthesized in the cells (p50gag and gp160/120env) were localized predominantly in cell membranes; however, some amount of p50 was found in cell nuclei. Thin-section immunoelectron microscopy showed accumulation of viruslike particles undistinguished from immature HIV-1 virions in the culture medium of the cells infected with vC5. The similar particles containing gag and env proteins were produced into the culture medium when the cells were coinfected with vC5 and vE234L strains. The particles contained heterogeneous cellular RNA, but no virus-specific RNA as shown by Northern blot hybridization. Immunization of the rabbits with purified viruslike particles produced virus-specific antibodies against gag and env proteins. The titer of antibodies was significantly higher than after immunization with cell lysate or recombinant proteins purified from the infected cells. Highly immunogenic HIV-1-like particles containing gag and env proteins but no virus-specific RNA are good candidates for potential vaccine.     

Chimeric gag-V3 virus-like particles of human immunodeficiency virus induce virus-neutralizing antibodies.

A 41-kDa unprocessed human immunodeficiency virus 2 (HIV-2) gag precursor protein that has a deletion of a portion of the viral protease assembles as virus-like particles by budding through the cytoplasmic membrane of recombinant baculovirus-infected insect cells. We have constructed six different combinations of chimeric genes by coupling the truncated HIV-2 gag gene to the neutralizing domain (V3) or the neutralizing and the CD4 binding domains (V3+CD4BD) of gp120 env gene sequences from HIV-1 or HIV-2. The env gene sequences were inserted either into the middle of the gag gene or at the 3' terminus of the gag gene. Virus-like particles were formed by chimeric gene products only when the env gene sequences were linked to the 3' terminus of the gag gene. Insertion of env gene sequence in the middle of the gag gene resulted in high-level chimeric gene expression but without the formation of virus-like particles. Three different chimeric genes [gag gene with HIV-1 V3 (1V3), gag gene with HIV-2 V3 (2V3), and gag gene with HIV-2 V3+CD4BD (2V3+CD4BD)] formed virus-like particles that were secreted into the cell culture medium. In contrast, the HIV-1 V3+CD4BD/HIV-2 gag construct did not form virus-like particles. The chimeric gag-env particles had spherical morphology and the size was slightly larger than that of the gag particles, but the chimeric particles were similar to the mature HIV particles. Western blot analysis showed that the gag-env chimeric proteins were recognized by antibodies in HIV-positive human serum and rabbit anti-gp120 serum. Rabbit anti-gag 1V3 and anti-gag 2V3 sera reacted with authentic gp120 of HIV-1 and HIV-2, respectively, and neutralized homologous HIV infectivity. Our results show that precursor gag protein has potential as a carrier for the presentation of foreign epitopes in good immunological context. The gag protein is highly immunogenic and has the ability to carry large foreign inserts; as such, it offers an attractive approach for HIV vaccine development.     

Production and characterization of simian--human immunodeficiency virus-like particles

We have produced and characterized, in a baculovirus expression system, simian-human immunodeficiency virus-like particles (SHIV VLPs) containing SIV Gag and HIV envelope (Env) proteins. Recombinant SIV gag (SIVmac239) and full-length or cytoplasmic domain-truncated HIV env from either HIV BH10 or HIV 89.6 virus were coexpressed in insect cells and Env incorporation into released SHIV VLPs was characterized. The expression level of the Env protein was found to be about 20-50% higher in both strains producing the truncated Env. Cell surface expression of the truncated Env proteins was found to be about eightfold higher than that of the full-length Env proteins. Furthermore, the truncated Env proteins exhibited higher levels of cleavage into gp120 and gp41 compared with the full-length Env. The SHIV VLPs produced by the coexpression of SIV gag and truncated HIV env contained both precursor (gp160) and gp120, while predominantly gp160 was found in the VLPs containing full-length Env. Coinfection of a recombinant virus expressing the protease furin also resulted in more efficient cleavage of gp160 to gp120. Both full-length and truncated Env were found to induce CD4+ cell fusion. Analysis of VLPs by immunoelectron microscopy demonstrated the incorporation of both full-length and truncated Env on the surface of VLPs. Truncated Env also was incorporated at higher levels on the surfaces of VLPs than full-length Env. The assembly of VLPs containing biologically active Env proteins may be useful in vaccine development and in functional studies of the HIV envelope protein.     

Human monoclonal antibody against a gag-coded protein of human immunodeficiency virus produced by a stable EBV-transformed cell clone

An EBV-transformed lymphoblastoid B cell clone (A12) derived from peripheral blood lymphocytes of an HIV-1-infected individual is described. The immunoglobulin isotype produced by this clone was IgM, and Southern blot analysis of immunoglobulin gene rearrangement showed a monoclonal pattern. The A12 monoclonal antibody was specific for the p24 product of the HIV-1 gag gene. This clone is now in continuous culture for more than 8 months and no changes in its biologic properties have been observed.     

Nef from pathogenic simian immunodeficiency virus is a negative factor for vaccinia virus

The nef gene of human and simian immunodeficiency viruses (HIV and SIV) is important for pathogenicity and maintenance of high virus loads. We previously reported that recombinant vaccinia viruses (rVVs) expressing nef from attenuated SIVmac1A11 (vNef1A11) produced typical plaques on thymidine kinase-deficient 143B cells, whereas rVVs expressing nef derived from the pathogenic SIVmac239 (vNef157) formed plaques with altered morphology. Here, we show that vNef157 is attenuated in normal and nude mice, whereas the pathogenicity of vNef1A11 is similar to that of a control virus. Thus, Nef157 is an attenuating factor in the vaccinia virus (VV) system, contrasting sharply with its function in lentiviruses. We also show that Nef157 inhibits VV cell-to-cell spread, causing formation of atypical plaques regardless of thymidine kinase deficiency, neoplasticity, and species of the infected cell line. We hypothesized that Nef157 interferes with VV spread by association with actin, but no direct colocalization of Nef and the cytoskeletal actin network was detected. Instead, higher levels of Nef157 protein were observed, although mRNAs for both nef genes were produced at comparable levels. Thus, the mechanism behind such Nef157 protein accumulation and Nef157-mediated VV attenuation could be related to the process that causes an opposite effect in its native SIV system, making SIVmac239 more pathogenic than SIVmac1A11.     

Transfer of the inducible lac repressor/operator system from Escherichia coli to a vaccinia virus expression vector.

Cis- and trans-acting elements of the Escherichia coli lac operon were transferred to vaccinia virus and used to regulate gene expression. A recombinant virus that constitutively expresses a modified lac repressor gene (lacI) was constructed. We calculated that each infected cell contained approximately 2 x 10(7) active repressor molecules (and 1-2 x 10(4) copies of the vaccinia virus genome). A strong vaccinia-virus late promoter was modified by insertion of the lac operator (lacO) at various positions. The ability of each modified promoter to regulate expression of beta-galactosidase was tested by transient assays in cells infected with wild-type or lacI-containing vaccinia virus. Placement of the lacO just downstream of the conserved TAAAT sequence of a late promoter was consistent with a minimal effect on basal expression and good repressibility, whereas basal expression was severely inhibited when lacO overlapped or preceded the TAAAT motif. A single recombinant vaccinia virus containing lacI and the beta-galactosidase gene under control of the optimal lacO promoter was constructed. In the absence of inducer, cells infected with this double recombinant virus synthesized little or no detectable beta-galactosidase. Addition of isopropyl beta-D-thiogalactoside restored expression to greater than 20% of the unrepressed level. This inducible vector system has potential applications for expression of heterologous and homologous genes.     

Effective tumor immunotherapy directed against an oncogene-encoded product using a vaccinia virus vector.

We have constructed a vaccinia virus recombinant that expresses the extracellular domain of the rat neu oncogene-encoded protein, a 185-kDa transmembrane glycoprotein termed p185. Strain NFS mice immunized with this recombinant virus developed a strong antibody response against the neu oncogene product and were fully protected against subsequent tumor challenge with neu-transformed NIH 3T3 cells. No tumor immunoprotection was found when recombinant virus-immunized mice were challenged with Ha-ras-transformed NIH 3T3 cells. These data indicate that immunization with a single oncogene-encoded antigen can fully and specifically protect animals against tumor cells bearing this antigen.     

Ebola virus-like particles protect from lethal Ebola virus infection

The filovirus Ebola causes hemorrhagic fever with 70-80% human mortality. High case-fatality rates, as well as known aerosol infectivity, make Ebola virus a potential global health threat and possible biological warfare agent. Development of an effective vaccine for use in natural outbreaks, response to biological attack, and protection of laboratory workers is a higher national priority than ever before. Coexpression of the Ebola virus glycoprotein (GP) and matrix protein (VP40) in mammalian cells results in spontaneous production and release of virus-like particles (VLPs) that resemble the distinctively filamentous infectious virions. VLPs have been tested and found efficacious as vaccines for several viruses, including papillomavirus, HIV, parvovirus, and rotavirus. Herein, we report that Ebola VLPs (eVLPs) were immunogenic in vitro as eVLPs matured and activated mouse bone marrow-derived dendritic cells, assessed by increases in cell-surface markers CD40, CD80, CD86, and MHC class I and II and secretion of IL-6, IL-10, macrophage inflammatory protein (MIP)-1alpha, and tumor necrosis factor alpha by the dendritic cells. Further, vaccinating mice with eVLPs activated CD4+ and CD8+ T cells, as well as CD19+ B cells. After vaccination with eVLPs, mice developed high titers of Ebola virus-specific antibodies, including neutralizing antibodies. Importantly, mice vaccinated with eVLPs were 100% protected from an otherwise lethal Ebola virus inoculation. Together, our data suggest that eVLPs represent a promising vaccine candidate for protection against Ebola virus infections and a much needed tool to examine the genesis and nature of immune responses to Ebola virus.     

Human immunodeficiency virus tat gene transfer to the murine central nervous system using a replication-defective herpes simplex virus vector stimulates transforming growth factor beta 1 gene expression.

The high incidence of neurological disorders in patients afflicted with acquired immunodeficiency syndrome (AIDS) may result from human immunodeficiency virus type 1 (HIV-1) induction of chemotactic signals and cytokines within the brain by virus-encoded gene products. Transforming growth factor beta1 (TGF-beta1) is an immunomodulator and potent chemotactic molecule present at elevated levels in HIV-1-infected patients, and its expression may thus be induced by viral trans-activating proteins such as Tat. In this report, a replication-defective herpes simplex virus (HSV)-1 tat gene transfer vector, dSTat, was used to transiently express HIV-1 Tat in glial cells in culture and following intracerebral inoculation in mouse brain in order to directly determine whether Tat can increase TGF-beta1 mRNA expression. dSTat infection of Vero cells transiently transfected by a panel of HIV-1 long terminal repeat deletion mutants linked to the bacterial chloramphenicol acetyltransferase reporter gene demonstrated that vector-expressed Tat activated the long terminal repeat in a trans-activation response element-dependent fashion independent of the HSV-mediated induction of the HIV-1 enhancer, or NF-kappaB domain. Northern blot analysis of human astrocytic glial U87-MG cells transfected by dSTat vector DNA resulted in a substantial increase in steady-state levels of TGF-beta1 mRNA. Furthermore, intracerebral inoculation of dSTat followed by Northern blot analysis of whole mouse brain RNA revealed an increase in levels of TGF-beta1 mRNA similar to that observed in cultured glial cells transfected by dSTat DNA. These results provided direct in vivo evidence for the involvement of HIV-1 Tat in activation of TGF-beta1 gene expression in brain. Tat-mediated stimulation of TGF-beta1 expression suggests a novel pathway by which HIV-1 may alter the expression of cytokines in the central nervous system, potentially contributing to the development of AIDS-associated neurological disease.     

Human immunodeficiency virus endocrinopathy

Human immunodeficiency virus (HIV) endocrinopathy encompasses a broad spectrum of disorders. Almost all the endocrine organs are virtually affected by HIV infection. HIV can directly alter glandular function. More commonly secondary endocrine dysfunction occurs due to opportunistic infections and neoplasms in immunocompromised state. The complex interaction between HIV infection and endocrine system may be manifested as subtle biochemical and hormonal perturbation to overt glandular failure. Antiretroviral therapy as well as other essential medications often result in adverse endocrinal consequences. Apart from adrenal insufficiency, hypogonadism, diabetes and bone loss, AIDS wasting syndrome and HIV lipodystrophy need special reference. Endocrinal evaluation should proceed as in other patients with suspected endocrine dysfunction. Available treatment options have been shown to improve quality of life and long-term mortality in AIDS patients.     

Intracisternal virus-like particles in a human pituitary adenoma.

Electron microscopy revealed the presence of intracisternal virus-like particles in a mixed pituitary adenoma consisting of growth hormone cells and prolactin cells. The tumor was removed by surgery from a 48 year old man with a multiple endocrine adenomatosis type I syndrome. The significance of virus-like particles in the pathogenesis of pituitary adenomas remains to be elucidated.     

Human immunodeficiency virus on the web: a guided tour.

Through the efforts of thousands of individuals, the World Wide Web has become a gold mine of information about HIV. In this article, we describe approximately 90 Web sites that are among the most useful to clinicians and researchers with regard to HIV. Web sites were classified according to their content and target audience and were judged according to their adherence to accepted standards of medical Internet publishing. Selected Web sites were categorized into the following groups: (1) sites with comprehensive coverage of HIV treatment and its management, (2) on-line peer-reviewed journals, (3) proceedings of scientific meetings, (4) sites with HIV-related textbooks, manuals, and guidelines, (5) government publications, (6) research databases, (7) information on clinical trials, (8) sites with comprehensive information for laypersons, and (9) sites with information related to specific medical complications of HIV infection.     

Human immunodeficiency virus tat-activated expression of poliovirus protein 2A inhibits mRNA translation.

To study the effect of poliovirus protein 2A on cellular RNA translation, the tat control system of human immunodeficiency virus (HIV) was used. Protein 2A was expressed from a plasmid construct (pHIV/2A) incorporating the HIV long terminal repeat. Protein synthesis was measured by using chloramphenicol acetyltransferase as a reporter gene driven by the Rous sarcoma virus long terminal repeat. When HIV/2A was cotransfected with the reporter, addition of a tat-producing plasmid caused at least a 50-fold drop in chloramphenicol acetyltransferase synthesis. A HeLa cell line carrying HIV/2A was established. In it, tat expression caused more than a 10-fold drop in chloramphenicol acetyltransferase synthesis from the reporter plasmid. Furthermore, 2A induction by tat caused cleavage of the cellular translation factor P220, a part of eukaryotic translation initiation factor 4F. Thus protein 2A can, by itself, carry out the inhibition of cellular protein synthesis characteristic of a poliovirus infection. Also, the HIV tat activation provides a very effective method to control gene expression in mammalian cells.     

Human immunodeficiency virus causes mononuclear phagocyte dysfunction.

There is compelling clinical evidence for dysfunction of the mononuclear phagocyte system in patients with AIDS, which is believed due in part to loss of T-cell cooperativity. The direct consequences of human immunodeficiency virus infection on macrophage function are unknown. To address this question we infected normal human macrophages in vitro with a monocytotropic strain of human immunodeficiency virus and performed assays to quantify their extra- and intracellular killing ability. Human immunodeficiency virus-infected macrophages were significantly less effective than control cells in mediating antibody-dependent cell-mediated cytotoxicity against leukemic cell targets and intracellular killing of Candida pseudotropicalis. The functional defects were profound, related temporarily to active virus production by the macrophages, and could not be overcome by granulocyte-macrophage colony-stimulating factor. Treatment of macrophages with 3'-azido-3'-deoxythymidine (AZT) 6 days after infection caused a marked decrease in virus production and prevented development of the intracellular killing functional defect. The results suggest that early antiviral therapy may be useful in preventing or mitigating some virus-induced mononuclear phagocyte dysfunction.     

Human immunodeficiency virus and opportunistic ocular infections.

Opportunistic ocular infections are a common and important complication of the acquired immunodeficiency syndrome. Cytomegalovirus retinitis is by far the most frequent infection, but other viral, bacterial, and parasitic infections of the posterior segment of the eye may be seen. Infections of the anterior segment are less common but may cause visual loss if not treated. Prompt recognition of these diseases is essential because ocular involvement may be an early sign of systemic dissemination.     

Attenuated Mengo virus as a vector for immunogenic human immunodeficiency virus type 1 glycoprotein 120.

Introduction of a sequence encoding 147 amino acids from human immunodeficiency virus type I (HIV-1) strain MN glycoprotein gp120 into the RNA genome of the stably attenuated Mengo virus strain vM16 yielded an infectious recombinant virus, vMLN450, which expressed the heterologous HIV-1 sequence along with the normal Mengo virus proteins. The HIV-1 gp120 sequence, fused to the amino terminus of the short, nonstructural Mengo virus leader polypeptide was recognized by a gp120 V3 loop-specific monoclonal antibody. When inoculated into mice, recombinant virus vMLN450 elicited a high-titer anti-HIV-1 antibody response as well as an HIV-1MN-specific cytotoxic cellular immune response. An anti-HIV-1 antibody response could also be detected in cynomolgus monkeys after a single immunization. We propose that attenuated Mengo virus can serve as an effective expression vector in cell systems and various animal species and offers another approach to the development of new, live recombinant vaccines.