Chimaeric HIV-1 subtype C Gag molecules with large in-frame C-terminal polypeptide fusions form virus-like particles

Halsey, Richard J.; Tanzer, Fiona L.; Meyers, Ann E.; Pillay, Sureshnee; Lynch, Alisson; Shephard, Enid G.; Williamson, Anna‐Lise; Rybicki, Edward P.

doi:10.1016/j.virusres.2008.01.012

Cited by 24 publications

(37 citation statements)

References 38 publications

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“…VLP of numerous virus types have been produced using the BEV-insect cell system for structural studies (9,48) or vaccines (20,28,42). In addition, recombinant AAV produced in insect cells has been used for preclinical and clinical studies (43,47).…”

Section: Discussionmentioning

confidence: 99%

Evidence of Prior Exposure to Human Bocavirus as Determined by a Retrospective Serological Study of 404 Serum Samples from Adults in the United States

Cecchini

Negrete

Virág

et al. 2009

Clin Vaccine Immunol

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Recently, molecular screening for pathogenic agents has identified a partial genome of a novel parvovirus, called human bocavirus (HBoV). The presence of this newly described parvovirus correlated with upper and lower respiratory tract infections in children. Lower respiratory tract infections are a leading cause of hospital admission in children, and the etiological agent has not been identified in up to 39% of these cases. Using baculovirus expression vectors (BEVs) and an insect cell system, we produced virus-like particles (VLPs) of HBoV. The engineered BEVs express the HBoV capsid proteins stoichiometrically from a single open reading frame. Three capsid proteins assemble into the VLP rather than two proteins predicted from the HBoV genome sequence. The denatured capsid proteins VP1, VP2, and VP3 resolve on silver-stained sodium dodecyl sulfatepolyacrylamide gels as three bands with apparent molecular masses of 72 kDa, 68 kDa, and 62 kDa, respectively. VP2 apparently initiates at a GCT codon (alanine) 273 nucleotides downstream from the VP1 start site and 114 nucleotides upstream from the VP3 initiation site. We characterized the stable capsids using physical, biochemical, and serological techniques. We found that the density of the VLP is 1.32 g/cm 3 and is consistent with an icosahedral symmetry with approximately a 25-nm diameter. Rabbit antiserum against the capsid of HBoV, which did not cross-react with adeno-associated virus type 2, was used to develop enzyme-linked immunosorbent assays (ELISAs) for anti-HBoV antibodies in human serum. Using ELISA, we tested 404 human serum samples and established a range of antibody titers in a large U.S. adult population sample.Among the family Parvoviridae, the genus Parvovirinae has many pathogenic species such as feline panleukopenia virus (38, 46), canine parvovirus (39), and Aleutian disease virus of mink (7). However, the only human-pathogenic parvovirus is the sole member of the Erythrovirinae, strain B-19 (3, 26, 52). Recently, a second potentially pathogenic human parvovirus was isolated and assigned the species named human bocavirus (HBoV) (2). Although HBoV DNA was detected in clinical isolates of children with lower respiratory tract infections (4,5,12,30), it is unclear whether HBoV was the etiological agent or contributed to the pathogenicity of the respiratory infection (34, 45). Until recently, the presence of HBoV in respiratory secretions relied on PCR (13,16,32,35,37,40). Using PCR, HBoV DNA has been detected worldwide with 5 to 10% prevalence among children with upper or lower respiratory tract infections (6,22,24,27,33,36,41,51). However, 80% of the HBoV DNA-positive patients were coinfected with common human respiratory viruses (8,10,17). Thus, whatever role HBoV plays in lower respiratory tract infection remains unclear.Using the baculovirus expression vector (BEV) system, we produced virus-like particles (VLPs) of HBoV. Two recent reports developed enzyme-linked immunosorbent assays (ELISAs) using HBoV antigen that associated into a homomeric...

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Section: Discussionmentioning

confidence: 99%

Evidence of Prior Exposure to Human Bocavirus as Determined by a Retrospective Serological Study of 404 Serum Samples from Adults in the United States

Cecchini

Negrete

Virág

et al. 2009

Clin Vaccine Immunol

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“…12 Development of plant-based subunit vaccines, although successful, was stopped as a result of low yields, and replaced with insect cell produced virus-like particle vaccine candidates (VLPs). The group investigated fusion proteins based on Gag, 14 and subsequently the optimising of production parameters and testing of immunogenicity of Gag-RT and Gag-Tat-Nef fusion VLPs. 15 Novel HIV vaccine vectors were also explored, with an enhanced experimental DNA vaccine for HIV being made that utilises an expression enhancer derived from an animal circovirus.…”

Section: Early Pipeline Hiv-1 Subtype C Vaccinesmentioning

confidence: 99%

South African HIV-1 vaccine candidates – the journey from the bench to clinical trials

et al. 2012

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“…6,7 Specifically, BEVS are beneficial for the production of protein-based vaccines because baculoviruses have a restricted host range (meaning no harmful effect on humans), insect cells are easy to handle and can be infected at a high multiplicity of infection (MOI), and recombinant proteins can be produced in large amounts, of a high quality and at a low cost. 5,8,9 However, as with other expression systems, the production of a given recombinant protein using BEVS is dependent on several variables. Factors such as insect cell line, cell density, MOI, infection time, medium type, temperature, and shaking speed have all been shown to affect the quality and quantity of recombinant protein expression.…”

Section: Introductionmentioning

confidence: 99%

“…The VLP constructs GagRT and GagTN consist of the highly conserved HIV-1 Gag polypeptide precursor (Pr55 gag ) fused with one or more inactivated regulatory (non-structural) proteins of HIV-1. 8 The GagRT construct encodes Gag and reverse transcriptase (RT) (Figure 1a), whereas the GagTN construct encodes Gag and a TatNef (TN) fusion protein (Figure 1b). The additional HIV-1 proteins Nef, Tat, and RT were included in the respective VLP vaccine candidates because they were previously identified as primary target regions for T-cell recognition in HIV-infected individuals in southern Africa.…”

Section: Introductionmentioning

confidence: 99%

“…8 Both VLP constructs have previously been demonstrated to express intact VLPs; however, their chimeric nature resulted in the formation of VLPs that were consistently larger than wild-type HIV-1 Gag VLPs (typically 100-140 nm in diameter). 8 In general, previous research has noted an inversely proportional relationship between chimeric VLP size and yields obtained.…”

Section: Introductionmentioning

confidence: 99%

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Optimization of chimeric HIV‐1 virus‐like particle production in a baculovirus‐insect cell expression system

Pillay

Meyers

Williamson

et al. 2009

Biotechnology Progress

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A baculovirus-insect cell expression system potentially provides the means to produce prophylactic HIV-1 virus-like particle (VLP) vaccines inexpensively and in large quantities. However, the system must be optimized to maximize yields and increase process efficiency. In this study, we optimized the production of two novel, chimeric HIV-1 VLP vaccine candidates (GagRT and GagTN) in insect cells. This was done by monitoring the effects of four specific factors on VLP expression: these were insect cell line, cell density, multiplicity of infection (MOI), and infection time. The use of western blots, Gag p24 ELISA, and four-factorial ANOVA allowed the determination of the most favorable conditions for chimeric VLP production, as well as which factors affected VLP expression most significantly. Both VLP vaccine candidates favored similar optimal conditions, demonstrating higher yields of VLPs when produced in the Trichoplusia ni Pro insect cell line, at a cell density of 1 x 10(6) cells/mL, and an infection time of 96 h post infection. It was found that cell density and infection time were major influencing factors, but that MOI did not affect VLP expression significantly. This work provides a potentially valuable guideline for HIV-1 protein vaccine optimization, as well as for general optimization of a baculovirus-based expression system to produce complex recombinant proteins.

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Chimaeric HIV-1 subtype C Gag molecules with large in-frame C-terminal polypeptide fusions form virus-like particles

Cited by 24 publications

References 38 publications

Evidence of Prior Exposure to Human Bocavirus as Determined by a Retrospective Serological Study of 404 Serum Samples from Adults in the United States

Evidence of Prior Exposure to Human Bocavirus as Determined by a Retrospective Serological Study of 404 Serum Samples from Adults in the United States

South African HIV-1 vaccine candidates – the journey from the bench to clinical trials

Optimization of chimeric HIV‐1 virus‐like particle production in a baculovirus‐insect cell expression system

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