Heparin-binding capacity of the HIV-1 NEF-protein allows one-step purification and biochemical characterization

Kohleisen, Birgit; Gaedigk-Nitschko, Kerstin; Ohlmann, Marion; Götz, Evi; Ostolaza, Helena; Goñi, Félix M.; Erfle, Volker

doi:10.1016/0166-0934(96)02049-6

Cited by 15 publications

(7 citation statements)

References 29 publications

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“…Protein immunization. Eight-to 12-week-old BALB/c, DBA/2, CBA, C57BL/6, and F 1 hybrid mice were immunized subcutaneously with recombinant protein Nef (20), Rev, or Tat in doses ranging from 5 to 50 g of protein in Freund or Ribi adjuvant (Sigma, St. Louis, Mo.). The mice were given booster immunizations 3 weeks after the primary immunization with the same antigen dose as in the primary immunization.…”

Section: Methodsmentioning

confidence: 99%

Recognition of prominent viral epitopes induced by immunization with human immunodeficiency virus type 1 regulatory genes

Hinkula

Svanholm

Schwartz

et al. 1997

J Virol

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Mice immunized with the regulatory genes nef, rev, and tat from human immunodeficiency virus type 1 developed both humoral and cellular immune responses to the gene products Nef, Rev, and Tat. This study demonstrates that it is feasible to induce immune reactions to all of these regulatory gene products. Humoral responses were seen after DNA boosts, while potent T-cell proliferative responses were noted already after a single immunization. A Th1-directed immune response was demonstrated early after immunization. A 3-to 75-fold-stronger T-cell response was seen in animals receiving DNA epidermally compared to that in animals receiving intramuscular injections. Nef, Rev, and Tat putative Band T-cell epitopes were clearly mapped by using peptides derived from the regulatory proteins and were similar to those which are detected in human immunodeficiency virus infection. Although immunization by the Nef, Rev, and Tat proteins raised high immunoglobulin G titers in serum, the epitope spreading appeared broader after DNA immunization. The combination of all of these regulatory genes together with two genes for structural proteins, the envelope and gag genes, demonstrated that a combined approach is feasible in that reactivities to all antigens persisted or were even augmented. No interference between plasmids was noted.

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Section: Methodsmentioning

confidence: 99%

Recognition of prominent viral epitopes induced by immunization with human immunodeficiency virus type 1 regulatory genes

Hinkula

Svanholm

Schwartz

et al. 1997

J Virol

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“…The antigens used were affinity-purified recombinant proteins Tat (kindly provided by Dr. Karn, Medical Research Council, UK) and Nef (kindly provided by Drs. B. Kohleisen and V. Erfle, GSF-National Research Center for Environment and Health, Neuherberg, Germany) [Kohleisen et al, 1996]. Recombinant Rev (Swedish Institute for Infectious Disease Control) expressed in a baculovirus system was also used.…”

Section: Immunoassaysmentioning

confidence: 99%

Genetic Immunization with Multiple HIV-1 Genes Provides Protection against HIV-1/MuLV Pseudovirus Challenge in vivo

Hinkula

Rollman²,

Lundholm³

et al. 2004

Cells Tissues Organs

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Superinfection by HIV-1 of a cell line containing the complete murine leukemia virus (MuLV) genome was shown to give rise to pseudotyped HIV-1/MuLV. Such superinfection was successful with certain strains of HIV-1 subtypes A–D. Primary spleen cells and cells of the peritoneal cavity of immunocompetent mice of the C57Bl/6 strain were infectable with the pseudotype HIV-1/MuLV and secreted HIV-1 in vitro and in vivo. In contrast, the murine cell lines, NIH 3T3, myeloma cell line Sp2/0, and two murine hybridoma cell lines were relatively resistant to infection and produced no or little HIV. After primary murine spleen cells had been infected with pseudotyped HIV-1 and transferred to C57Bl/6 mice, replication-competent HIV-1 was obtained from the peritoneal cavity for at least 10–14 days. High amounts (>10⁵ vRNA copies/ml) of HIV-1 vRNA could be measured in the peritoneal fluid. Presence of HIV-1 proviral DNA was detectable in cells from the peritoneal cavity for up to 24 days after infected cell transfer. Active reverse transcriptase representing both HIV-1 and C-type murine retroviruses was detected in the peritoneal washes. The HIV-infected spleen cells injected into the peritoneal cavity elicited HIV-1-specific cellular immune responses to p24gag, gp160Env, Nef, Tat and Rev. Mice immunized with HIV-1 DNA, but not with HIV-1 protein, cleared their HIV-1-infected cells within 10–14 days after challenge with HIV-1/MuLV-infected syngeneic spleen cells. This novel model system of primarily cellular reactivity to HIV-1-infected cells in vivo may become useful for assaying experimental HIV-1 immunization schedules.

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“…Both p27 and p25 isoforms have been expressed in different biological systems such as E. coli (Azad et al 1994 ), yeast (Macreadie et al 1998 ; Sirén et al 2006 ), insect cells (Kohleisen et al 1996 ), mammalian cells (Cooke et al 1997 ) and plant cells (Marusic et al 2007 ; Barbante et al 2008 ; de Virgilio et al 2008 ; Zhou et al 2008 ; McCabe et al 2008 ).…”

Section: Nef Protein As a Plant-made Hiv Vaccine Componentmentioning

confidence: 99%

Plant-based strategies aimed at expressing HIV antigens and neutralizing antibodies at high levels. Nef as a case study

et al. 2009

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The first evidence that plants represent a valid, safe and cost-effective alternative to traditional expression systems for large-scale production of antigens and antibodies was described more than 10 years ago. Since then, considerable improvements have been made to increase the yield of plant-produced proteins. These include the use of signal sequences to target proteins to different cellular compartments, plastid transformation to achieve high transgene dosage, codon usage optimization to boost gene expression, and protein fusions to improve recombinant protein stability and accumulation. Thus, several HIV/SIV antigens and neutralizing anti-HIV antibodies have recently been successfully expressed in plants by stable nuclear or plastid transformation, and by transient expression systems based on plant virus vectors or Agrobacterium-mediated infection. The current article gives an overview of plant expressed HIV antigens and antibodies and provides an account of the use of different strategies aimed at increasing the expression of the accessory multifunctional HIV-1 Nef protein in transgenic plants.

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Heparin-binding capacity of the HIV-1 NEF-protein allows one-step purification and biochemical characterization

Cited by 15 publications

References 29 publications

Recognition of prominent viral epitopes induced by immunization with human immunodeficiency virus type 1 regulatory genes

Recognition of prominent viral epitopes induced by immunization with human immunodeficiency virus type 1 regulatory genes

Genetic Immunization with Multiple HIV-1 Genes Provides Protection against HIV-1/MuLV Pseudovirus Challenge in vivo

Plant-based strategies aimed at expressing HIV antigens and neutralizing antibodies at high levels. Nef as a case study

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