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Methods and compositions comprising bacteriophage nanoparticles

A composition and phage technology, applied in the field of T4 phage, can solve the problems of inability to package DNA, inability to accurately determine the number of binding sites, inability to use excitation-enhancement strategies, etc., and achieve reliable production effects

Active Publication Date: 2011-09-21
CATHOLIC UNIV OF AMERICA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Given this variability in the polymeric head model, the number of binding sites available on the particle cannot be accurately determined using excessive experimentation
Thus, it would be very difficult, if not impossible, to control the copy number of foreign antigens on the aggregated head
In addition, because of their shape, the polymeric heads cannot package DNA and thus cannot be used in prime-boost strategies known in the art

Method used

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  • Methods and compositions comprising bacteriophage nanoparticles
  • Methods and compositions comprising bacteriophage nanoparticles
  • Methods and compositions comprising bacteriophage nanoparticles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Construction, overexpression and purification of p24-Hoc

[0087] A DNA fragment corresponding to the full-length p24 polypeptide (225 amino acids, 24 kDa) was ligated to the 5'-end of the hoc gene via the DNA sequence encoding the pro-gly-gly linker sequence. As mentioned above, p24 is the large capsid subunit of the HIV capsid, which encloses 2 molecules of the HIV genome and other protein (e.g., reverse transcriptase, integrase) and nucleic acid (e.g., tryptophan tRNA primer) components, It is necessary for infection. This can be achieved through the SOE strategy disclosed by Kuebler and Rao, 1998. In-frame insertion of the construct into the BamHI site of the T7 expression vector pET15b (Novagen Inc. Madison, WI, USA) resulted in the insertion of a 26 amino acid sequence consisting of a hexahistidine tag to the N-terminus of the p24-Hoc protein sequence. combined ( image 3 (A)). Induced by IPTG, the expressed 66kDa six His-p24-Hoc fusion protein accounts for ab...

Embodiment 2

[0089] In vitro assembly of T4 nanoparticles

[0090] To assemble or "load" recombinant antigen onto the surface of T4 phage particles, approximately 2 x 10 10 Sucrose gradient - purified HOC - soc - T4 nanoparticles with TMG buffer (50mM sodium phosphate buffer, pH7.0, 75mM NaCl and 1mMMgSO 4 ) with increasing amounts of purified HIV-p24-Hoc and incubated at 37° C. for about 60 min. Then, the resulting T4 nanoparticles were precipitated at 14,000 rpm for 60 min, and the unbound supernatant fraction was discarded. Wash the pellet twice with excess buffer to remove any unbound or non-specifically captured protein. All samples, starting material, unbound and bound fractions, and controls were analyzed by 4-20% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie blue. refer to Figure 4 , the proportion of HIV-p24-Hoc and Hoc binding sites is indicated on the top of the figure. Lanes are as follows: St, starting p24-Hoc; Su, p24-...

Embodiment 3

[0092] Specificity and stability of in vitro assembly systems

[0093] p24-Hoc and hoc - soc - The binding interactions between T4 nanoparticles are highly specific. Figure 5 explains this specificity. Using the experimental design of Example 2, T4 nanoparticles were incubated with p24 alone (lanes 2-4) or a mixture of p24 and p24-Hoc (lanes 5-7). When compared to control phage (lane 1, C-Ph), p24 bound to particles only when fused to Hoc (lanes 5-7). Note that no significant p24 binding occurs. The position of p24-Hoc is marked with an arrow. These results indicate that fusion to the Hoc polypeptide or a fragment thereof is required for binding to T4 particles. Neither the control proteins BSA (66 kDa) nor anthrax PA (89 kDa) showed significant binding to T4 particles (data not shown).

[0094] The stability of the interaction between displayed p24-Hoc and T4 phage particles was assessed by treating p24-T4 nanoparticles with pH 2.0 buffer or 6M urea, and whether eithe...

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Abstract

Compositions and methods comprising bacteriophages are provided. In particular, the present invention includes novel and customized T4 bacteriophages uniquely designed for effective antigen and foreign particle presentation. The present invention also provides in vitro methods for the making of customized T4 bacteriophages. The compositions and methods of the present invention may be used for effective vaccine delivery systems.

Description

field of invention [0001] The present invention relates to novel methods and compositions comprising bacteriophage. More specifically, the present invention includes novel and custom T4 phages uniquely designed for efficient antigen and foreign particle presentation. The methods and compositions of the invention can be used in efficient vaccine delivery systems. Background of the invention [0002] In phage display, foreign peptides, domains or proteins are fused to structural proteins and exposed on the outer surface of the phage capsid (Smith, 1985). The coat proteins (M13, fd, and fl), the small coat protein pIII (4–5 copies), and the large coat protein pVIII (2700 copies) of filamentous bacteriophage have been widely used to generate A combinatorial library of peptides from ® (Smith and Petrenko, 1997; Manoutcharian et al., 2001). Other display systems using icosahedral phages lambda and T7 have also been developed (Maruyama et al., 1994; Danner and Belasco, 2001). T...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/00C12Q1/70A01N63/00G01N33/53A61K39/07A61K39/12A61K39/21A61K39/295C07K14/16C12N7/00C12N15/10C12N15/86C12Q1/68C40B40/02
CPCA61K39/07A61K2039/64C12N2740/16222A61K2039/5256C12N2740/16234C07K2319/21C12N2795/10121C07K2319/735A61K39/21A61K2039/645C12N2795/10143C07K14/005C12N2740/16322C12N7/00A61K2039/6075C12N15/1037C40B40/02A61K2039/627C12N2740/16034A61K39/295C12N2740/16334C12N15/86A61K39/12A61K2039/55505A61K2039/57A61K2039/70A61P31/04A61P31/12A61P33/00A61P33/02A61P43/00C07K2299/00C07K2319/00
Inventor 文尼加拉·巴沙维斯瓦拉·劳
Owner CATHOLIC UNIV OF AMERICA
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