Vaccines for prevention and treatment of addiction
An addictive drug, adenovirus technology, applied in the field of 001, can solve problems such as unclear effectiveness of vaccine strategies
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Embodiment 1
[0086] This example demonstrates a method for eliciting an immune response in a mammal using an adenoviral vector-fluorophore conjugate.
[0087] The fluorophore Cy3 was conjugated to the capsid protein of an adenovirus serotype 5 vector (Cy3Ad). Cy3Ad was delivered to mice by intravenous administration, and the serum of mice was collected three weeks later.
[0088] Western blot was performed to determine the presence of anti-Cy3 antibodies in serum samples from immunized mice. Briefly, annexin or Cy3-conjugated annexin (Cy3-annexin) was loaded on SDS-PAGE gels. After electrophoresis, proteins were visualized by Coomassie brilliant blue staining, or transferred to nitrocellulose membranes, and western blot was performed using control mouse serum or Cy3Ad-immunized serum as primary antibody. Western blot results showed that the Cy3Ad-immunized mice contained Cy3-specific antibodies in the sera, but not in the PBS-injected control mice.
[0089] The results of this example s...
Embodiment 2
[0091] This example demonstrates that a humoral immune response can be elicited in mammals with an adenovirus containing a nicotine antigen conjugated to a capsid protein.
[0092]Mice were immunized with nicotine-conjugated Ad, or a mixture of unconjugated Ad and nicotine as a negative control. Two weeks after immunization, serum was collected and analyzed for nicotine-specific antibodies by protein immunoassay, and sera from immunized mice were used as primary antibodies to detect nicotine-conjugated target antigens or control antigens. Mice immunized with a mixture of unconjugated Ad and nicotine did not develop a humoral immune response against nicotine according to Western blot results. No antibodies reactive with the negative control antigens BSA or KLH were developed with nicotine-conjugated Ad. In contrast, sera from mice immunized with nicotine-conjugated Ad strongly reacted with nicotine-conjugated Ad, nicotine-conjugated BSA, and nicotine-conjugated KLH according t...
Embodiment 3
[0097] This example describes the generation of adenovirus-nicotine conjugates.
[0098] In a series of experiments, two restricted nicotine analogs (i.e., compounds 1 and 2 of Meijler et al, J.Am.Chem.Soc, 125:7164-7165 (2003) shown below) were synthesized and added A linker (below, "R") generates "CNA" "CNI" as follows:
[0099]
[0100] Furthermore, in a non-limiting nicotine analogue, namely, trans-S'-hydroxymethylnicotine (#H948175, Toronto Research Compounds, North Yorkshire, Ontario, Canada), a linker is added via the hydroxyl group to thereby to allow cross-linking of proteins. All three haptens were conjugated to BSA for assay experiments, to KLH or ovalbumin for attachment to 40 nm polystyrene beads, and to capsid protein for immunization studies. Individual products of organic synthesis were obtained on a Bruker AMX-600 (600 MHz), AMX-500 (500 MHz), or AMX-400 (400 MHz) spectrometer 1 H NMR spectra obtained on a Bruker AMX-500 (125.7 MHz) or AMX-400 (100.6 MHz...
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