HIV TAT-CD4 hybried molecules and methods of use thereof
A CD4, hybrid technology, applied in the direction of hybrid peptides, chemical instruments and methods, botany equipment and methods, etc., can solve the problems that antibodies cannot provide antiviral effect, host immune system is reduced, etc.
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Embodiment 1
[0317] Embodiment 1 designs the hybrid protein containing TAT polypeptide and CD4 minimum module
[0318] A new hybrid protein containing the human CD4env binding domain was designed as follows. analyzed the three-dimensional (3D) structure of CD4-gp120 and identified regions of CD4 involved in env binding. Specifically, the region corresponding to CD4 amino acid residues 15-85 is substantially completely buried in the CD4-binding pocket of env (Fig. 4). This fragment is known as the CD4 minimal module and is stabilized by the presence of a disulfide bond between Cys16 and Cys84.
[0319] The CD4 minimal assembly (residues 15-85) was then cloned into various expression cassettes to express the Tat-CD4 fusion protein using standard molecular biology techniques. The expression cassette comprises a sequence encoding the CD4 minimal module flanked by a sequence encoding a Tat polypeptide. Specifically, as shown in Figure 5, the expression cassette includes from 5' to 3' directi...
Embodiment 2
[0323] Example 2 Preparation of ENV-fusion protein complex
[0324] Stable purified env-hybrid protein complexes were prepared with and without formaldehyde treatment. For example, to induce a conformational change in env, equimolar concentrations of env (such as SF2 or SF162) and a hybrid molecule containing the CD4 minimal module and one or more Tat polypeptides are incubated together for 1 hr at 37°C. At the cellular level, these interactions are transient. Thus, at the end of the incubation, half of the complexes were fixed with formaldehyde while the other half remained untreated. Treated and untreated complexes were separated on a Superdex-200 column. Purified fractions were analyzed on HPLC columns and SDS-PAGE. This purified complex contains both env and fusion proteins. Furthermore, these complexes are expected to be homogeneous and will contain no more than 2-3% free fusion protein.
[0325] The ability of Env to complex with hybrid molecules was monitored by in...
Embodiment 3
[0326] Example 3 produces neutralizing antibodies
[0327] Vaccines comprising compositions of the invention can be delivered by a variety of routes well known in the art, including, for example, delivery of polypeptide antigens and / or delivery of polynucleotides expressing the polypeptides in one or more formulations. For example, a DNA prime / protein boost strategy allowed screening in rabbits and nonhuman primates for the ability of multiple Env constructs to present epitopes in situ in the host when delivered as DNA vaccines. DNA vaccination may involve administration of naked DNA, DNA complexed to microparticles (such as PLG particles), or DNA as part of a viral vector, followed by a protein boost. Electroporation or DNA vaccination using viral vectors, among other methods described herein, can be used to efficiently deliver polynucleotides encoding hybrid proteins and / or Env polypeptides to non-human mammals (eg, primates).
[0328] A. CD4-TAT hybrid protein-ENV complex ...
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