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Aug 22, 2021

COVID-19 Breakthrough:  T4 phage surface Covid-19 full-length spike protein vaccine oral immunization in ducks elicited immune response.

Oral administration of T4 bacteriophage nanoparticle surface displaying SARS-CoV-2 (COVID-19) full-length Spike protein as vaccine carrier in adult egg-laying ducks, elicited high antibody response detected by ELISA from blood serum. The results demonstrated the induced antibodies contained very low levels of antibody background from anti-T4 phage particle shells. In our study, the quantity of surviving T4 phage vaccine particles have been detected within blood, samples from all internal organ tissues, contents of the entire digestive tract, and excreted stool. Also, we conducted the T4 phage vaccine absorption, presence, distribution, and elimination kinetic data analysis. We found the full-length spike T4 phage vaccine to be non-toxic and harmless. Study includes breakthrough of full-length (1273-aa) coronavirus spike protein with natural trimeric conformation coded by 3822bp DNA expressed in E. coli bacteria.

Construction of T4 phage COVID-19 candidate vaccines with Spike subtype 

In this study we use T4 phage particle carrier displaying COVID-19 Spike protein in different recombinant structure T4 phage particles as vaccine-antigen, including T4-WHS, T4T7-S and T4-63. For duck serum antibody ELISA detection as the contrast antigen, we also use spike subunits with different lengths and different immunogenicity displayed on T4 phages surface as contrasts to observe duck antibodies induced by the full-length Spike protein vaccine T4-WHS and evaluate respective immunoaffinity differences. In phage T4-63, the RBD fragment is encoded in another ORF that avoids common mutants seen in COVID-19 Alpha, Beta, Gamma, Delta, and Omicron variants. Currently, subvariants of omicron are circulating, including BA.4, BA.5, and BA. 2.12.1, our candidate vaccine strain T4-63 also avoided these mutations.

Materials and methods

  • Bacteriophage T4 Gene-Protein Surface Display System (T4-GPSD)

  • Construction of T4 phage ФT4 Full-length Spike protein COVID-19 vaccine

  • Oral vaccine phage ФT4-full-length Spike purification 

  • Duck immunization and blood collection 

  • Quantifying vaccine T4 phage particles in blood and organs

  • Serum antibody ELISA determination 

Blood antibody titer vs date course 

The goal of this study was to evaluate in animal subjects the systemic immune response to oral vaccination of T4 phage vaccine and digestive-tract mucosal interaction. Results showed that on days 1, 23, and 37 antibodies were undetectable in blood samples (Figure 3). We speculate that these data illustrate the difference in antibody inducement via oral vaccination versus subcutaneous injection. With oral immunization, more days may be required for the large full-length Spike molecule carrying T4 phage particles to distribute in spleen and to generate the antibodies to reach a detectable titer, as measured on days 52 and 65. Further, it will be interesting to evaluate differences in induced antibody levels based on varying dosage frequencies and amounts, as well as evaluating in BALB/c mice.    


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