Industry Updates Volume 18

Covid 19 - Down, But Not Out

Despite the decision of many countries over the last two months to ease or eliminate COVID restrictions, it is clear that COVID is not over. There are a number of distinct themes:

• The WHO still has COVID-19 classified as a world-wide Pandemic - the world celebrated the 2-year anniversary of that designation during March 2022
• Many countries which have suddenly reopened, have experienced a surge in infections
  • This includes many European countries, the UK, as well as selected areas in the U.S.
  • The true extent of this may be hard to measure, as a number of countries, including the UK, cut back their testing programs shortly after re-opening. 
• A second factor is the rising prevalence of the BA.2 variant of COVID-19 (‘Stealth OMICRON)
  • The WHO decided not to consider this a separate variant of concern, but rather to count this together with the BA.1 version of OMICRON
  • This somewhat clouds the current trends, as we are having reports of people already infected with BA.1 getting another infection from BA.2
  • Somewhat tellingly, the FDA has halted its approval of the GlaxoSmithKline therapeutic Xevudy Sotrovimab, which appeared to work against BA.1, as it doesn’t appear to work against BA.2
  • Meanwhile the CDC estimates that half of all new infections in the U.S. are BA.2
• A separate theme is what is happening in countries that were previously largely-closed and where Omicron has now penetrated
  • The first case to note is Hong Kong, where infections rose dramatically from a very low base, but in the last week however, the infection rate in Hong Kong has dropped significantly
  • Death rate in Hong Kong from this surge has been surprisingly high and it has been attributed to a significant proportion of older people, perhaps as much as ⅓, deciding not to be vaccinated for fear of the COVID vaccines, combined with their age and comorbidities
  • As Hong Kong begins to regain control of the virus, a bigger question is China, where the major city of Shanghai and its 25 Million+ population is now under full lockdown - it has been estimated that as many as 130 Million 60+ year olds in China have not been vaccinated, given ‘vaccine hesitancy’ issues - similar to what was happening in Hong Kong

• Attached in the Appendix, Exhibits 1 and 2 show the period of domination of the Delta variant, which accounted for nearly 100% of infections worldwide, running from June 2021 to the end of December 2021
  • This is followed by the surge in Omicron infections in December and now causing nearly 100% of COVID infections worldwide
• Exhibit 3 shows infection rates over the most recent seven days in six countries
  • We see that while the infections in Hong Kong have fallen significantly, there has been a jump in infections in Germany, France, UK and Israel
  • It is not clear if this is simply a short term effect due to the reopening programs of the relevant countries, or if it is a new wave triggered by the transmissibility of the BA.2 variant
• ​​As for death rates over the last seven days, there has been a noticeable increase in Germany
• Infection and death rates continue to decline in the U.S., although there are regional variations

• A final perspective worth considering, are the cumulative trends across these countries in infections and deaths
  • Despite the enormous surge in Hong Kong over the last month, it is only at half the level, or less, of the other countries in cumulative terms of infections
  • Even less in terms of deaths - see Exhibit 4
    • Cumulative death rate in Hong Kong - @ 982 per million - is close to the COVID death rate in Israel @ 1,128 per million
    • Both cumulative death rates are much lower than United States @ 2,936 per million
    • Major European countries fall between these extremes
  • The significantly lower death rate (62% lower) in Israel compared to the U.S. appears to be due to the faster and higher take up of the initial vaccination campaign, followed subsequently by the third and in some cases fourth shots as boosters
  • It may also be influenced - as in Europe - by the greater accessibility of national health services for the entire population

Concerns:

• Are we seeing the start of a major BA.2 surge in Europe and elsewhere?
• Will China now face widespread COVID infections which it appears to have avoided so far?

Refana Update - Proof of Concept on WIV Vaccine Yields Positive Results

• We have tentatively named our vaccine RefanaVax-MV → MV = MultiValent
• In our ongoing Pre-Clinical study @ IITRI in Chicago, mice were prime/boost vaccinated
• Although vaccinated mice showed anti-Spike ELISA antibody titers, they also showed substantial neutralizing and ELISA titers after boost, against both the WA1/2020 and Delta variants (neutralizing titer of approximately 640 post-boost)
• Vaccinated mice remained completely healthy, no side-effects from the vaccines
• Protective efficacy of the vaccine was evaluated with a live SARS-CoV-2 challenge - a 10X ‘fatal’ dose of Delta virus → all the vaccinated mice survived
• Details follow - these results suggest that the Refana WIV vaccine design against SARS-CoV-2 is robust and worth further developing

Table 1 - Neutralizing Antibodies vs. Wuhan Strain

• Table 1 compares neutralizing antibody titres of the RefanaVax-VM Delta Variant COVID vaccine against the original Wuhan WA1 strain of COVID
• None of the three vaccinated animals - TA 1, TA 2, TA 3 - nor any of the three control animals - initially had a measurable titre
  • All < 40 at day 21 - 1st column
  • By day 42, after the vaccine booster shot, two of the three vaccinated animals - TA1 and TA3 - developed good neutralizing antibody titres - 160 and 452 respectively - 2nd column
  • These results compare with a titre of 1280 in the serum of a human who had recently received their third Pfizer vaccine booster shot

Table 2: Neutralizing Antibodies vs. Delta Strain

• Table 2 has same structure as Table 1, but now measuring results vs. the Delta variant
• As before, none of the control animals exhibited measurable neutralizing antibodies
• However, all three vaccinated animals did have strong responses to Delta
  • Two of the vaccinated animals - TA1 and TA3 - achieve a full threshold of the measurable titre in this experiment (= 640)
  • Second animal TA2 achieved 56.6
• In all cases, results achieved against the Delta variant are significantly stronger than what was achieved against original WA1 Wuhan variant
  • Not surprising, as the RefanaVax-MV Delta vaccine was designed using the whole Delta virion
  • This result is a mirror image of what occurred with the approved vaccines - all of which were designed against the original Wuhan variant and which subsequently showed lower levels of protection against the Delta variant
  • RefanaVax-MV, used here, is a Delta specific vaccine, achieving a much stronger result
• Our Chief Vaccine Advisors tells us that these results are about 3X better than what he has seen with other vaccine development Pre-Clinical studies

Table 3 - IGG antibody ELISA results

• Table 3 shows the overall IGG antibody results from an ELISA test
  
• Control animals had very negligible results on Day 21 - by Day 42 control animal number 1 had died from other causes, whereas the results from control animals 2 and 3 were still below a measurable range
  
• All vaccinated animals had considerable levels of antibodies by Day 21, but with a more than 5x from the highest and lowest results - first column
  
• The impact of the booster shot was to increase the IGG levels of all three animals significantly by between almost 5x to almost 9x - 2nd column
  

​Challenge Results

• All mice in the above study, both vaccinated with the Refana Delta specific COVID vaccine, and the unvaccinated control mice, were challenged with the live Delta virus
  
• IITRI had previously established ‘LD50’ levels with respect to mice being challenged with live COVID virus, which led to the death of 50% of the mice in that cohort
  
• This live challenge of the mice was at a viral content of approximately 10 times higher than the LD50 level - considered a ‘fatal’ dose
  
• This massive dose killed all the unvaccinated mice
  
• Vaccinated mice showed no symptoms whatsoever, including mouse TA2, which had exhibited very low neutralizing antibodies - as shown in the prior tables
  
  • Presumably, TA2 was protected by the wider protective effect of the Whole Virion Vaccine, possible via stimulating a t-cell response
    

IITRI Paper - American Society of Virology (ASV) Conference - Wisconsin

• See attached, Appendix 2, scientific paper abstract submitted to the ASV Conference
  
• Presented at their annual conference in July 2022
  
  • First in-person meeting in 3 years, given the Pandemic
    

Next Steps

Refana is now working with a number of parties on a funding proposal to NIAID, the National Institute of Allergy and Infectious Diseases, for next stages of funding, which would take this project, potentially, to full trials and if successful, ultimate approval

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Appendix 1

Exhibit 1 - Covid Cases per Million People - 7 day rolling average

Exhibit 2 - Covid Deaths per Million People - 7 day rolling average

Exhibit 3 - Cumulative Covid Cases per Million People

Exhibit 4 - Cumulative Covid Deaths per Million People

Exhibit 5 - Delta Variant

Exhibit 6 - Omicron Variant

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Appendix 2

Development of a Small Scale Whole Inactivated Vaccine Against B.1.617.2 SARS-CoV-2 Delta
Andrew Eaton1, Landon Westfall1, Dianet Giraldo1, Brian Maccaba2, Phillip Schwartz3 and Robert Baker1*

1 Affiliation 1: Division of Microbiology and Molecular Biology, Illinois Institute of Technology Research Institute (IITRI), Chicago, IL 60616
2 Affiliation 2: Refana™, Middletown, DE 19709
3 Affiliation 3: EnteraBio, Jerusalem Israel 9112002
*Correspondence: rbaker@iitri.org

Vaccine development against SARS-CoV-2 variants in humans will be benefited from using multiple types of vaccine constructs. Whole inactivated virus (WIV) vaccines are safe and have been used in the prevention of respiratory viruses such as influenza and polio as well as others, and have the added benefit of more stable storage. Here we have produced, inactivated, purified, and concentrated a Delta B.1.617.2 WIV and tested its efficacy using K18-hACE2 heterozygous mice. We compared varying growth conditions including serum-containing and serum-free medias. Total protein was quantified in both medias with values of 927 µg/mL and 2 µg/mL respectively. Following optimization of conditions, Vero E6 cells were grown in a Corning HyperFLASK® and infected with SARS-CoV-2 Delta (B.1.617.2). After harvest, virus was concentrated 500-fold using centrifugal filters and quality controlled at multiple steps using qPCR, Bradford, and SDS-PAGE. The original volume, prior to concentration, had a total SARS-CoV-2 genome content at 1.20E+11 copies. Following 100-fold concentration the total genomes copies was 3.78E+10 which correlates to a ~69% loss during processing. The final vaccine preparation was prepared with a squalene-in-water emulsion adjuvant and tested in K18-hACE2 mice  along with shame-vaccinated controls. Mice were prime/boost vaccinated with a total of 0.2 µg and 1.9 µg S1 RBD, respectively. Although vaccinated mice showed only anti-Spike ELISA antibody titers, they showed substantial neutralizing and ELISA titers after boost against both the WA1/2020 and Delta variants (neutralizing titer of approximately 640 post-boost). Protective efficacy of the vaccine was evaluated with a live SARS-CoV-2 Delta challenge.  These findings suggest that using a whole inactivated vaccine against SARS-CoV-2 should be further evaluated. A whole inactivated vaccine tend to be more temperature stable to reduce transport costs and associated issues to mitigate logistical challenges in delivering to other countries.
 
Key words:
Delta B.1.617.2, Whole Inactivated Virus Vaccine, Neutralization titers

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