Examining the interactions of Galahad™ compound with viruses to develop a novel inactivated influenza A virus vaccine

Abstract

Galahad™ is a proanthocyanidin complexed with polysaccharides that inactivates viruses and indicates potential for an innovative approach to making protective vaccines. The polysaccharide portion of Galahad™ consists mainly of arabinan and arabinogalactan. In a seven-day toxicity study in rats, it was not toxic even when tested undiluted. Galahad™ inactivated a wide range of DNA and RNA viruses including adenoviruses, corona viruses such as SARS-CoV-2, and influenza viruses. Electron microscopy studies showed that exposure to Galahad™ caused extensive clumping of virions followed by lack of detection of virions after longer periods of exposure. Based on the viral inactivation data, the hypotheses tested is that Galahad™ inactivation of virus can be used to formulate a protective inactivated virus vaccine. To evaluate this hypothesis, infectious influenza A virus (H5N1, Duck/MN/1525/81) with a titer of 10^5.7 CCID₅₀/0.1 ml was exposed for 10 min to Galahad™. This treatment caused the infectious virus titer to be reduced to below detectable limits. The Galahad™ -inactivated influenza preparation without adjuvant or preservative was given to BALB/c mice using a variety of routes of administration and dosing regimens. The most protective route of administration and dosing regimen was when mice were given the vaccine twice intranasally, the second dose coming 14 days after the primary vaccine dose. All the mice receiving this vaccine regimen survived the virus challenge while only 20% of the mice receiving placebo survived. This suggests that a Galahad™-inactivated influenza virus vaccine can elicit a protective immune response even without the use of an adjuvant. This technology should be investigated further for its potential to make effective human vaccines.

Keywords: Catechin; H5N1; Influenza; Polysaccharide; SARS-CoV-2 (COVID-19); Vaccine.

Figure 1

Electron microscopic examination of the effects of Galahad™ (200 μg/mL) treatment on the structure of influenza virus-like particles. A. Influenza VLPs without treatment, shown at a high magnification. B. VLPs at 2.5 minutes after exposure to Galahad™ shown at high magnification. C. VLPs at 12 minutes after exposure to Galahad™ shown at high magnification. D. Percentage of clumped particles observed (black bars), micrographs without particles (gray), and particles per micrograph compared to those in the untreated experiment (white bars). Views of particles are similar to those seen in micrographs shown in panels A–C and were used to count the particles to calculate the percentages shown in panel D. E. VLPs untreated (left), Galahad™-treated for 2.5 minutes (middle), and Galahad™-treated for 12 minutes (right), shown at a medium magnification.

Figure 2

Survival of BALB/c Mice Immunized with Galahad™ Inactivated Vaccines. A. Vaccine 1: 10-minute exposure to Galahad™ with vaccine given once intranasally to BALB/c mice and subsequently challenged with 10^3.5 TCID50 influenza A H5N1 (Duck/MN/1525/81). 10^5.7 TCID₅₀∗P < 0.05. 10^4.7 TCID₅₀ ∗P < 0.05. 10^3.7 TCID₅₀ ∗P < 0.05. No Vaccine (PSS). B. Vaccine 2: 10-minute exposure to Galahad™ with vaccine given twice intranasally to BALB/c mice and subsequently challenged with 10^3.5 TCID50 influenza A H5N1 (Duck/MN/1525/81). 10^5.7 TCID₅₀ ∗∗∗P < 0.001. 10^4.7 TCID₅₀ ∗∗∗P < 0.001. 10^3.7 TCID_50.. No Vaccine (PSS). C. Vaccine 3 a 24-hour exposure to Galahad™ with vaccine given twice intranasally to BALB/c mice and subsequently challenged with 10^3.5 TCID50 influenza A H5N1 (Duck/MN/1525/81). Mice received vaccine 2 and 3 at 28 and 14 days before virus challenge; vaccine 1 14 days before virus challenge. 10^5.7 TCID₅₀ ∗∗∗P < 0.001. 10^4.7 TCID₅₀ ∗∗∗P < 0.001. 10^3.7 TCID₅₀. No Vaccine (PSS).

Figure 3

The Effect of Galahad™-Inactivated Vaccines on Lung Virus Titers after Infectious Virus Challenge. A. Vaccine 1: 10-minute exposure to Galahad™ with vaccine given once intranasally to BALB/c mice and subsequently challenged with 10^3.5 TCID50 influenza A H5N1 (Duck/MN/1525/81). B. Vaccine 2: 10-minute exposure to Galahad™ with vaccine given twice intranasally to BALB/c mice and subsequently challenged with 10^3.5 TCID50 influenza A H5N1 (Duck/MN/1525/81). C. Vaccine 3: 24-hour exposure to Galahad™ with vaccine given twice intranasally to BALB/c mice and subsequently challenged with 10^3.5 TCID50 influenza A H5N1 (Duck/MN/1525/81). Mice received vaccine 2 and 3 at 28 and 14 days before virus challenge; vaccine 1 14 days before virus challenge. ^5.7 TCID₅₀ ∗∗∗P < 0.001. ^4.7 TCID_50.^3.7 TCID_50.No Vaccine (PSS).

Figure 4

Virus Neutralizing Antibody Titers Measured Relative to Time of Infectious Virus Challenge (10^3.5 TCID50). A. Vaccine 1: 10-minute exposure to Galahad™ with vaccine given once intranasally to BALB/c mice and subsequently challenged with 10^3.5 TCID50 influenza A H5N1 (Duck/MN/1525/81). B. Vaccine 2: 10-minute exposure to Galahad™ with vaccine given twice intranasally to BALB/c mice and subsequently challenged with 10^3.5 TCID50 influenza A H5N1 (Duck/MN/1525/81). C. Vaccine 3: 24-hour exposure to Galahad™ with vaccine given twice intranasally to BALB/c mice and subsequently challenged with 10^3.5 TCID50 influenza A H5N1 (Duck/MN/1525/81). ^aThe inverse of the most dilute serum sample completely protecting cells from virus cytopathic effects was considered the virus neutralization titer for the serum. ^bDetectable limit of the assay refers to lowest dilution used. Serum was diluted by a factor of 1/100 for use in the neutralizing antibody assay. ^cMice were so dehydrated, that it was not possible to obtain virus from samples. Mice received vaccine 2 and 3 at 28 and 14 days before virus challenge; vaccine 1 14 days before virus challenge. ^5.7 TCID₅₀. ^4.7 TCID_50.^3.7 TCID_50.No Vaccine (PSS).

Figure 5

Lung Function as Measured by Saturated Oxygen (SaO₂) Levels at Day 7 after Virus Challenge. A. Vaccine 1: 10-minute exposure to Galahad™ with vaccine given once intranasally to BALB/c mice and subsequently challenged with 10^3.5 TCID50 influenza A H5N1 (Duck/MN/1525/81). B. Vaccine 2: 10-minute exposure to Galahad™ with vaccine given twice intranasally to BALB/c mice and subsequently challenged with 10^3.5 TCID50 influenza A H5N1 (Duck/MN/1525/81). C. Vaccine 3: 24-hour exposure to Galahad™ with vaccine given twice intranasally to BALB/c mice and subsequently challenged with 10^3.5 TCID50 influenza A H5N1 (Duck/MN/1525/81). Mice received vaccine 2 and 3 at 28 and 14 days before virus challenge; vaccine 1 14 days before virus challenge. ∗P < 0.05. ^5.7 TCID₅₀. ^4.7 TCID_50.^3.7 TCID_50.No Vaccine (PSS).

Figure 6

Lung Pathology Scores in BALB/c Mice Immunized with Galahad™ Vaccines A. Vaccine 1: 10-minute exposure to Galahad™ with vaccine given once intranasally to BALB/c mice and subsequently challenged with 10^3.5 TCID50 influenza A H5N1 (Duck/MN/1525/81). B. Vaccine 2: 10-minute exposure to Galahad™ with vaccine given twice intranasally to BALB/c mice and subsequently challenged with 10^3.5 TCID50 influenza A H5N1 (Duck/MN/1525/81). C. Vaccine 3: 24-hour exposure to Galahad™ with vaccine given twice intranasally to BALB/c mice and subsequently challenged with 10^3.5 TCID50 influenza A H5N1 (Duck/MN/1525/81). Mice received vaccine 2 and 3 at 28 and 14 days before virus challenge; vaccine 1 14 days before virus challenge. ∗P < 0.05, ∗∗P < 0.01,∗∗∗P < 0.001. ^5.7 TCID₅₀. ^4.7 TCID_50.^3.7 TCID_50.No Vaccine (PSS).