CFI HIV Therapeutic Vaccine Technology

Using a purely physical method called CFI for effectively inactivating HIV while maintaining protein integrity and antigenicity to generate a humoral response

Direct CFI Treatment Is Necessary and Sufficient To Inactivate HIV-1

Experiments were performed to determine the necessity of exposing the virus directly to the supercritical N2O/CO2. Culture media was run through the CFI unit under conditions that inactivate HIV. This culture media was used to dilute HIV in the infectivity assay and the TCID50 of this virus was compared to HIV that was directly exposed to supercritical N2O/CO2. Only HIV that was directly run through the CFI unit was inactivated.CFI-HIV-Vaccine CFI-Inactivation-of-HIV-Graph sm

To determine if high pressure alone could inactivate HIV, the virus was pressurized to 3,000 psig for 15 minutes (this time exceeds CFI exposure) in the CFI unit and then removed. We also examined whether N2O/CO2 at a non-supercritical pressure could inactivate HIV by exposing the virus to N2O/CO2 in the CFI unit at 500 psig. Less than 0.5 log inactivation was obtained in both experiments.

This observation is also supported by data from other investigators, who reported that much higher pressures and residence times are needed to inactivate HIV and SIV by hydrostatic pressure alone.

Major HIV Structural Proteins Are Present After CFI Treatment

CFI-HIV-Vaccine Western-Blot-of-CFI-Treated-HIV-Proteins smDetermination of the viral proteins after CFI treatment was performed by Western blot. Untreated viral stocks and CFI-treated samples were separated on a 4-12% Bis-Tris gel (Invitrogen) and then transferred to nitrocellulose.

Murine monoclonal antibodies to four structural proteins were used to detect gag- and env-encoded proteins, gp160 (Chessie 8; ARRRP), gp41 (Zeptometrix), p24 (Zeptometrix), and p17 (Zeptometrix). All of these proteins were present in CFI-treated and untreated controls in similar quantities.

This experiment also provided evidence that these proteins are still antigenic after CFI treatment and can be recognized by a panel of antibodies.

CFI Compromises HIV Virion Structure

To explore the possibility of a compromised virion structure, the density of CFI-treated virions was determined. HIV has a density range of 1.16 to 1.18 g/mL; a density of 1.17 g/mL was measured for the untreated virus (data not shown). CFI-treated virus, where complete inactivation was achieved, resulted in a density of 1.05 g/mL, suggesting a compromised structure that may include punctures in the virion. A similar density was observed for CFI-treated virions where 3.4 logs of virus were inactivated.

CFI-HIV-Vaccine Density-of-CFI-Treated-HIV-Graph sm

CFI-Treated HIV is Capable of Generating a Humoral Immune Response

Each mouse was injected with 1.8 μg of p24. On days 0, 14, 21 and 28 post inoculation retro-orbital bleeds were performed and the sera collected. Antibody titers were measured against a purified HIV lysate in a standard ELISA. Positive (anti-gp41, Chessie 8) and negative controls (normal mouse serum; NMS) were included with each plate. The reciprocal of the highest dilution that gave a positive result, as determined by the mean plus two standard deviations of the negative control, was taken as the antibody titer.

In cases where only one replicate was positive, the dilution was considered positive only if the average of the replicates fell above the cutoff. The titer of antibodies was similar when mice were inoculated with either heat-treated or CFI-treated HIV (t Test p= 0.49). Therefore, CFI-treated HIV is capable of generating a humoral immune response in mice.

Advantages and Benefits CFI HIV Therapeutic Vaccine Technology

  • CFI treatment specifically inactivates more than 5 logs of HIV in a single-stage unit (reaching the threshold of detectable infectivity).
  • CFI treatment can theoretically inactivate more than 30 logs of HIV in a 5-stage unit in a single step, continuous flow process.
  • While viral proteins are retained and were shown to be antigenic after CFI treatment of HIV-1, the virion structure is compromised as demonstrated by its altered sedimentation pattern.
  • The vaccine potential of the CFI technique rests with the phenomenon of incomplete destruction of virus particles with complete inactivation.
  • CFI inactivation results in the preservation of a majority of HIV protein domains that are necessary for the induction of an efficient immune response.
  • CFI treatment that causes incomplete destruction of the HIV virion results in the exposure of cryptic internal epitopes.
  • HIV strains of different clades or immunotypes may be used to provide greater safety and broader protection.
  • CFI HIV therapeutic vaccine technology is inexpensive, amenable to large-scale processing, and can be readily implemented in a host country site.

Partnering and Collaboration Opportunities

We are seeking strategic corporate partners and/or international research institutions in developing a CFI HIV therapeutic vaccine technology and vaccine.

We are also interested in collaborating with strategic corporate partners and/or international research institutions in developing CFI vaccines for aqua-cultured fish as well as farm animals and household pets.

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