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. 2009 Sep;46(15):2911-7.
doi: 10.1016/j.molimm.2009.07.008. Epub 2009 Aug 8.

Preclinical Evaluation of Innate Immunity to Baculovirus Gene Therapy Vectors in Whole Human Blood

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Free PMC article

Preclinical Evaluation of Innate Immunity to Baculovirus Gene Therapy Vectors in Whole Human Blood

Lindsay J Georgopoulos et al. Mol Immunol. .
Free PMC article

Abstract

Interactions of gene therapy vectors with human blood components upon intravenous administration have a significant effect on vector efficacy and patient safety. Here we describe methods to evaluate these interactions and their effects in whole human blood, using baculovirus vectors as a model. Opsonisation of baculovirus particles by binding of IgM and C3b was demonstrated, which is likely to be the cause of the significant blood cell-associated virus that was detected. Preventing formation of the complement C5b-9 (membrane attack) complex maintained infectivity of baculovirus particles as shown by studying the effects of two specific complement inhibitors, Compstatin and a C5a receptor antagonist. Formation of macroscopic blood clots after 4h was prevented by both complement inhibitors. Pro- and anti-inflammatory cytokines Il-1beta, IL-6, IL-8 and TNF-alpha were produced at variable levels between volunteers and complement inhibitors showed patient-specific effects on cytokine levels. Whilst both complement inhibitors could play a role in protecting patients from aggressive inflammatory reactions, only Compstatin maintained virus infectivity. We conclude that this ex vivo model, used here for the first time with infectious agents, is a valuable tool in evaluating human innate immune responses to gene therapy vectors or to predict the response of individual patients as part of a clinical trial or treatment. The use of complement inhibitors for therapeutic viruses should be considered on a patient-specific basis.

Figures

Figure 1
Figure 1
Complement activation by baculovirus as determined by ELISA for C3a (A) and C5b-9 (B). Each data point represents the mean of three volunteers and the error bars represent 1 standard deviation. Filled squares = virus; open squares = 10-fold diluted virus; triangles = cell debris; crosses = PBS; open circles = virus plus Compstatin (Compst).
Figure 2
Figure 2
(A) Presence of IgM antibodies that bind to BV particles in human serum as shown using ELISA. G10 is the medium in which BV is suspended. Bars represent the mean of three volunteers and the error bars represent 1 standard error of the mean. (B) Presence of BV particles in complex with c3b in the serum of volunteer C as detected by a combined ELISA and PCR method, using an immobilised capture antibody that binds C3b followed by BV-specific PCR. (C) Location of virus particles in separated whole blood as shown by BV-specific PCR. M=mononuclear cell fraction, E=erythrocyte fraction, P=plasma, Compst. = Compstatin.
Figure 3
Figure 3
(A) Circulating cytokine levels 4h post-inoculation with baculovirus and control samples as detected using the CBA human inflammation kit (BD Biosciences) in the presence and absence of complement inhibitors. Levels of IL-10 and IL-12p70 were undetectable in all samples. Black bars represent data from volunteer A, spotted bars from volunteer B and striped bars from volunteer C. (B) Mean cytokine levels from all three volunteers normalised to the neat virus samples. White bars represent loops containing PBS, grey bars represent 10-fold diluted virus, striped bars represent cell debris and black bars represent virus.
Figure 4
Figure 4
Blood clotting was assessed over time. (A) Free platelets present in the blood were counted. Large squares = virus; small squares = 10-fold diluted virus; triangles = cell debris; crosses = PBS; open circles = virus plus Compstatin (Compst.) and filled circles = virus plus C5aRA. (B) Production of thrombin:antithrombin (TAT) complexes was measured using ELISA. Grey bars represent 10-fold diluted virus; black bars = undiluted virus; vertical striped bars = cell debris; spotted bars = PBS; diagonal striped bars = virus plus Compstatin (Comps) and white bars = virus plus C5aRA. The TAT assay failed for volunteer C.

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