Multiplex RT-PCR amplification of HIV genes to create a completely autologous DC-based immunotherapy for the treatment of HIV infection |
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Authors: | Tcherepanova Irina Harris Jason Starr Aijing Cleveland Jaclyn Ketteringham Helen Calderhead David Horvatinovich Joe Healey Don Nicolette Charles A |
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Institution: | Research and Development Department, Argos Therapeutics, Inc., Durham, North Carolina, USA. itcherepanova@argostherapeutics.com |
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Abstract: | BackgroundEffective therapy for HIV-infected individuals remains an unmet medical need.
Promising clinical trials with dendritic cell (DC)-based immunotherapy
consisting of autologous DC loaded with autologous virus have been reported,
however, these approaches depend on large numbers of HIV virions to generate
sufficient doses for even limited treatment regimens.Methodology/Principal FindingsThe present study describes a novel approach for RT-PCR amplification of HIV
antigens. Previously, RT-PCR amplification of autologous viral sequences has
been confounded by the high mutation rate of the virus which results in
unreliable primer-template binding. To resolve this problem we developed a
multiplex RT-PCR strategy that allows reliable strain-independent
amplification of highly polymorphic target antigens from any patient and
requires neither viral sequence data nor custom-designed PCR primers for
each individual. We demonstrate the application of our RT-PCR process to
amplify translationally-competent RNA encoding regions of Gag, Vpr, Rev and
Nef. The products amplified using this method represent a complex mixture of
autologous antigens encoded by viral quasispecies. We further demonstrate
that DCs electroporated with in vitro-transcribed HIV RNAs
are capable of stimulating poly-antigen-specific CD8+ T cell
responses in vitro.Conclusion/SignificanceThis study describes a strategy to overcome patient to patient viral
diversity enabling strain-independent RT-PCR amplification of RNAs encoding
sequence divergent quasispecies of Gag, Vpr, Rev and Nef from small volumes
of infectious plasma. The approach allows creation of a completely
autologous therapy that does not require advance knowledge of the HIV
genomic sequences, does not have yield limitations and has no intact virus
in the final product. The simultaneous use of autologous viral antigens and
DCs may provoke broad patient-specific immune responses that could
potentially induce effective control of viral loads in the absence of
conventional antiretroviral drug therapy. |
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