Introduction: SMARTube is offering a new approach to serological diagnostics of infectious diseases – a major public health issue. False positives, false negatives and inability to determine stage of infection represent a true handicap in accurately diagnosing hundreds of millions of people annually and thus constitute a major unmet medical need.

The company develops unique products called SMARTube™, which are blood sample processing devices for improving specificity and sensitivity of serological assays for infections such as: HCV, HIV, COVID19, HBV, XMRV, CMV, HTLV-1, Chagas, Tuberculosis, Zika, Ebola, Borrelia and others, reducing the health costs and the spread of the diseases by identifying false positive and false negative diagnosed patients in hundreds of millions tests a year.

The Problem: Current serological assays suffer from non-specific reactions leading to high false positives, indicating on infection while it does not exist. On the other hand, the assays suffer from a relatively low sensitivity, unable to detect infections weeks or months until the antibodies against the viruses (such as HCV, HIV and others) reach a detectable level (the "window period"). In addition, it is impossible for current serological assays to distinguish between a false positive, recent or chronic infection and viral cleared/resolved infection. Thus, current methods require repeat serological tests, in addition to the virus direct nucleic acid testing (NAT) causing unnecessary expenses and loss of valuable time that is needed for proper therapeutic and other clinically related decisions.

The SMARTube™: The SMARTube is a blood sample processing device containing a proprietary activation media for the stimulation of antibody production (In-Vitro). It is a complementary product for improving performance of standard serological assays by reducing the rate of both false positive and false negative results. SMARTube enables detection of infections during the "window period" (when the standard serology is negative), enables validation of a positive infection and also effectively distinguishes between various stages of infection: acute, chronic, active or viral cleared/resolved infection (be it spontaneous or drug elicited), while the standard serological test cannot produce this information.

The Technology: The SMARTube is based on the company's proprietary platform technology, Stimmunology™ allowing the growth and differentiation of primed lymphocytes (sensitised lymphocytes that have been exposed to the virus) that are present in the patient's whole blood sample. It includes an activation media in which whole blood lymphocytes are stimulated to produce virus specific antibodies in-vitro within days. The Stimmunology is overcoming the immune suppression of the body and thus, antibodies which were not present or detected in the original sample would be present and detectable in the enhanced samples following the Stimmunology incubation.

The Products: SMARTube has developed three products: SMARTube™HCV, SMARTube™HIV and combined SMARTube™HIV&HCV. The company conducted clinical trials with these products in various countries around the world (such as USA, Israel, Russia, China, South Africa, Kenya, Mexico and Confidential 3 others). The results of these clinical trials have been published in both peer-reviewed journals and international conferences. The products' effectiveness was clinically proven in various serological assays, enabling its integration with any serological assay. The SMARTube™ HIV&HCV has received the CE mark as well as regulatory clearance in several other countries. The SMARTube™HIV is currently in regulatory clearance process with the US Food and Drug Administration ("FDA"). SMARTube works in collaboration with prominent academic institutes such as the University of Maryland and New York University as well as with the U.S Military and other governmental health organizations

Competitive Landscape: Traditional testing of an infectious disease (such as HCV) begins with either a rapid or a laboratoryconducted assay for detection of antibodies in the blood via serology test. The laboratory serology tests are usually automated. The device and kits market is controlled by global major players such as Abbott, Roche and Siemens. The competition in serology tests is focused on the sensitivity and the need to reduce false negatives. These companies have improved the detection from the first generation to the current fourth generation and increased the sensitivity by detecting virus antigen in the same serology test. However, this sensitivity increases the false positives and causing a burn costs to healthcare system because about 50% of the positives are false. All the positives need to be re-tested and if found to be positive in the second test, a direct Nucleic Acid Test (NAT) will be performed. NAT is expensive compared to serology tests, 100-300$ per test as compared to approx. 20$ for serology. By adding the SMARTube to serology tests, the companies will be able to reduce the high rate of false positives and also increase their diagnostic resolution by calculating the stimulation index (SI) that indicates the stage of infection. To the best of our knowledge, here is no other technology in the market that can identify in one step the stage of infection. A one step analysis will save the cost of shipping second blood samples from patients and re-analyzing them, and save millions of NAT expensive and unnecessary testing of false or viral cleared positives. In monitoring HCV-positive patient, the cost-effectiveness of using SMATube is even greater. Those patients are monitored routinely by NAT to identify viral clearance as the current serology tests can not indicate clearance. Analyzing these patients with SMARTube will save the expensive NAT tests for an estimated 20 million people in the western world that are routinely tested every 6 month.

Unmet Need
Approximately one third of the world's population is influenced by infectious diseases accounting for a quarter of the overall 56 million deaths annually. Current gold standard for infectious disease diagnostics is serological testing (immunoassays) which attempts to detect virus specific antibodies, produced by the immune system, in a blood sample. Testing for infections caused by viruses, such as Hepatitis C (HCV), begins with either a rapid or a laboratory-conducted assay for HCV antibodies in the blood via serology test. The laboratory serology tests are automated and devices and kits are controlled by global major players such as Abbott, Roche, Siemens and others. The competition in serology tests is focused on the sensitivity and the need to reduce false negatives. These companies improved the detection from the first generation to the current fourth generation and increased the sensitivity by improving binding and by detecting virus antigen in the same serology test. However, this sensitivity increases the false positives and causing a burn costs to healthcare system because a large percent of tested people are found to be negative eventually (false or cleared), but are analyzed repeatedly via serology and NAT to confirm that they are negative instead of being diagnosed at the first test.

In 2015, the global demand for immunoassays was about $17 billion of which over 35% was a demand for infectious disease immunoassays (see table below). The infectious disease immunoassay market continues to develop with global demand expecting to increase at a five-year compound annual growth rate (CAGR) of 4.6%. Worldwide growth has the potential to expand to nearly $7.5 billion by 2017.

However, current serology encounters several challenges in accurately diagnosing hundreds of millions of people. Described below are the main challenges in which our SMARTube products and patented platform technology is offering a solutions:

1. Determining stage of infection
Confidential 5 Current serology does not enable distinction between various stages of an infection (active, resolved, acute, chronic, etc'). In HCV, for example, 15-25% of those infected will "spontaneously" clear the virus while antibodies will remain in their blood stream for some time after clearance. Thus, a positive serological result cannot indicate whether there is an active infection, a crucial indication necessary to initiate treatment. Currently, costly direct viral Nucleic Acid Testing (NAT) must be performed following a positive serological result in order to determine if an HCV infection is currently active. However, since HCV is a hepatic virus and does not always reside in the blood in detectable levels, viral detection can be missed.

In addition, current serology cannot distinguish acute from chronic HCV infection. Such distinction has important clinical impact as it has been found that initiating treatment during the acute stage offers substantial benefits and may reduce significantly the development of chronic disease.

2. Post-therapy Monitoring
One of the main utilities of infectious disease diagnostics is the referral of infected patients to therapy. Therapies are very costly (approx. $50,000/year per patient in HCV) and require continuous monitoring to analyze the effectiveness of the drug and to ensure viral clearance. These patients will be observed and monitored years after infection, in fact the rest of their life, in case the infection becomes active again. Approx. 50% of HCV treated patients will have reoccurrence after completion of therapy because of phenomena called "viral escape" – the virus is not active, escaped into organs and is not detectable in blood. Current viral monitoring of these patients is done using viral direct NAT methods that are very costly (approx. $100/test) and may not always detect the virus as it may not reside in the blood stream or be below detection levels of the assay. Serology tests are not indicative in these patients as they can be positive for long time after infection and the current serology tests cannot identify the viral clearance.

3. False Negatives:
Usually, when an infectious agent or any other foreign structure (antigen) enters the body, the cells of the immune system recognize it, are primed by it, and with the help of the general immune system, end up producing antibodies against that pathogen/antigen. This whole process takes only a few days, and antibodies are found in measurable levels in the blood within 5-7 days after infection/exposure. However, in various infections (such as HCV, HIV and others) this is not so. The serological Window Period (a.k.a. Seroconversion Time) is the time between exposure/infection and the time when there are measurable amounts of antibodies in the blood. Therefore, if a serological test is taken during the Window Period the result will be negative since antibodies are not yet present at a detectable level. However, the infected person may transmit the virus to others during that period (through blood contact, sexual contact, organ/ tissue transplant, blood donations, etc').

Implications for the Window Period include the need for follow up repeat testing several months after the initial test. In addition, the inability to detect a person in the Window Period prevents that person from entering therapy and in certain cases (such as HIV in pregnant women) allows the transmission of HIV from the mother to the fetus while the probability for such transmission can be reduced from 30% to 1-2% with currently available treatment. Intensive effort have made to shorten the Window Period of the current tests, but still the Window Period, in HCV for example, is 6 months (97%) for current available serology with antigen and NAT detection shortening the Window Period by up to 50 and 60 days, respectively.

4. False Positives and Viral Cleared:
Current serological assays suffer from non-specific reactions and inability to identify viral clearance leading to high false positives, indicating on infection while it does not exist. Verifying the presence of an infection minimizes unnecessary medical visits and ensures that counseling, medical referral, and evaluation are targeted for patients confirmed as infected. In HCV, for example, of those found to be positive in an initial serology test, about 35% are found to be negative in a second serology test (false). One of the implications of such high false positivity is the need for repetitive serology testing. Furthermore, serology confirmed positives samples are analyzed further in a third test – by NAT, to identify viral presence directly (since the RIBA confirmatory assay is no longer available). This expensive test is performed in all serology positive samples. While 80% are diagnosed as HCV positives and need a treatment, about 20% of these serology positive patients are diagnosed as viral cleared, increasing dramatically the overall costs of the diagnostics process. These patients do not need any therapeutic action, but to be monitored twice a year as recommended. Thus, there is a clear need for a technology that lowers the serology rate of false/viral cleared positive results.

Our Target Markets

We identified commercialization opportunities for SMARTube in large target markets in which SMARTube clearly demonstrates added clinical and cost effective values as follows:

Hepatitis C (HCV) Testing Market
Hepatitis C is an infectious disease affecting primarily the liver, caused by the hepatitis C virus (HCV). Over 200 million people are infected worldwide with 4 million new infections every year. Approx 80% of newly infected individuals will progress to develop chronic infection leading to scarring of the liver and ultimately to cirrhosis and liver cancer. HCV is the most common blood borne virus in the US with approx. 4 million people infected and around 20,000 new infections annually. While HCV is Confidential 7 transferred primarily by blood-to-blood, the risk of prenatal transmission (mother to child) can be as high as 19% if co-infected with HIV.

Until recently, there were no effective drugs for HCV but the interferon-based therapies with major side effects and physicians tended to wait for a spontaneous viral clearance (~20% of cases) before starting treatment. Out of the 4 million people in the US infected with HCV, only about 100,000 people received treatment. Treating patients too late, in the chronic HCV phase, results in enormous health costs. The treatments of chronic HCV complications were estimated to reach over $10 Billion in the U.S alone by 2019.

But the market is facing a significant change from the old treatments to early diagnosis and new effective treatments of HCV. Recently FDA approved new therapies anticipated to reach the market soon (including oral administered drugs). These innovative drugs interfere directly with HCV replication with almost no side effects, driving the market for early diagnosis and treatment of HCV. Such treatment and diagnosis will reduce the epidemiology effect of HCV and the enormous costs of patients having complications of chronic HCV infection. One of indicators for such a change is the new CDC guidelines recommending screening all the population born between 1945 and 1965 (the baby boomers). In the US, these individuals represent about 27% of the population (approx. 86 million people) who need to be screened according to the new CDC guidelines and at a major interest of HCV pharmaceutical companies. About 75% of all HCV infected people in the US are part of this population segment.

The reason for screening the high risk population of the baby boomers is the gathering evidence of asymptomatic viral infection, viral escape in viral cleared HCV patients and their high probability to develop second and even more repeated viral infections. This is also the reason why all positive diagnosed HCV patients that were cured are still routinely monitored all their lives for HCV.

Processing a blood sample in the SMARTube and comparing antibody levels pre and post processing provides the Stimmulation Index which can indicate a reoccurrence as well as differentiate those with active infection from the 15-25% of people who spontaneously clear the virus yet still have HCV antibodies in their blood stream for a long time after clearance. In addition, since there is no cure for HCV (only therapy for reducing levels of virus), people treated need to be followed and monitored approx. every 6 months for the rest of their lives since about 50% of people after treatment have a reoccurrence.

The estimated market size for this application would be substantial as it is estimated that 2% of the western world population is infected with HCV (20 Million people) of which 75% are at a risk group of being born between 1945-1965 (15 Million people). This risk group is approx. 27% of the general population in the western world totaling 270 Million people who need to screened for HCV in order to detect those 15 million individuals infected within this population segment. In addition to screening 270 million people with the SMARTube (in order to distinguish active current from cleared infection), all those found to be positive would need to enter therapy and monitoring for the rest of Confidential 8 their lives using the SMARTube totaling 480 million – 1.3 billion tests (given monitoring every 6 months and life expectancy of 80 years).

In addition, the SMARTube can be used as an effective tool in testing additional high risk population for HCV such as healthcare workers who experience needle stick injuries (over 37 million incidents annually) or dialysis patients who have a very high prevalence and incidence of HCV (over 2 million patients screened several time a year).

Corona virus (COVID19)
Corona virus (COVID19) itself has been sequenced and its genome was assembled by international teams and helped in generating a reference strain that intended for use in viral detection assays. Infect, several groups have developed PCR-based tests to identify the virus which got the USA-FDA emergency use authorization (EUA). However, the virus level in the blood of infected people is very limited since it is up-taken by special white blood cells, the B-Cells and thus detection its presence by molecular way is facing a lot of obstacles. Thus, serology tests are required according to the new FDA-issued guidelines to identify the titer of the B-Cells that are proliferating upon confronting with the virus and release into the blood antibodies, initially the anti-COVID19 antibodies are IgM type and then IgG. Thus the serological test provides the identification of the stimulation of B-cells in the blood and the identification of the antibody group types and their prevalence.

The performance of these assays (sensitivity, specificity, positive and negative predictive values) when used in asymptomatic people are relatively low. Since most of people attend the test at asymptomatic stage, results should be interpreted with caution, and in the context of other available clinical and epidemiological information. The proposed project offers the use of SMARTube technology that has been previously proven effective and reliable for HIV and HCV testing. The SMARTube technology enables a rapid proliferation of B-Cells and increase of antibodies secretion required for the serology test. The special attribute of this tube is that it enables a rapid proliferation of the stimulated B-Cell and therefore enables a much better serological test of the viral infection to overcome the most common limitations of virus infections diagnosis: 1) the false negative results of antibody testing (due to the long time that usually takes for the B-Cell proliferation), 2) the false positive results due to cross reactive problems, and 3) the lack of virus presence in the blood at the time of testing. Thus we can expect significantly better analysis for serological testing with the SMARTube blood processing application as pre-analytical step for anti-COVID19 antibodies detection improving, coupled to a simple IgG/IGM ELISA immune-assay test.

Cytomegalovirus (CMV)
Infections with Cytomegalovirus (CMV), a member of the herpesvirus family, are common in man and are usually mild and asymptomatic. However, in pregnant women, newborns, and immunocompromised individuals CMV infections may pose a significant medical risk. CMV is the most common cause of intrauterine infection and the leading infectious cause of sensorineural Confidential 9 hearing loss and mental retardation in newborns. Transmission can occur after either primary or secondary infection, but the likelihood is much greater after primary infection, with a probability of 30% to 40%. The burden of disease for congenitally infected infants is high, with 10% to15% having symptoms at birth, including intrauterine growth restriction, microcephaly, hepatosplenomegaly, petechiae, jaundice, chorioretinitis, thrombocytopenia, and anemia. Of infants who are symptomatic at birth, 20% to 30% will die, and 90% of the symptomatic survivors will have late complications. Despite 85% to 90% of congenitally infected infants showing no signs or symptoms at birth, late sequelae appear in 5% to 15%. These include sensorineural hearing loss, delay of psychomotor development, and visual impairment. CMV infection remains difficult to diagnose on symptoms alone since a high percentage of infections remains asymptomatic. Since the risk of in utero virus transmission and CMV related damage of the fetus is markedly increased during primary infection, reliable recognition of primary CMV infection is of high importance for pregnant women.

One of the key components in prenatal diagnosis of congenital CMV infection is distinguishing between maternal primary and secondary infection, a major handicap for current serologic testing. Primary infection occurs in a seronegative person who has never been infected before. Following primary infection, the virus persists in a latent state. Secondary infection occurs when an individual with a history of primary infection has a reactivation of the latent virus. Since identifying early stage of infection has a major clinical impact and can influence the course of a pregnancy, market size for this application would include screening all pregnant women. There are 130 million births annually around the world with over 10 million births in the western world alone. In addition, it has been found that universal screening of pregnant women is cost effective in the western world.

Human Immunodeficiency Virus (HIV)
The Human Immunodeficiency Virus (HIV) targets the immune system and weakens people's surveillance and defense systems against infections and some types of cancer. As the virus destroys and impairs the function of immune cells, infected individuals gradually become immunodeficient. Immune function is typically measured by CD4 cell count. Immunodeficiency results in increased susceptibility to a wide range of infections and diseases that people with healthy immune systems can fight off. The most advanced stage of HIV infection is Acquired Immunodeficiency Syndrome (AIDS), which can take from 2 to 15 years to develop depending on the individual. AIDS is defined by the development of certain cancers, infections, or other severe clinical manifestations.

Confidential 10 Globally, approx. 35 million people are living with HIV/AIDS with 2.7 Million new infections annually. 2 million children are currently living with HIV/AIDS and every day, 1,000 infants acquire HIV during pregnancy, delivery and breastfeeding. In the U.S., over 1 million people are living with HIV/AIDS with a new HIV Infection taking place every 9.5 minutes.

One in five (21%) of people living with HIV is unaware of their infection and it is those unaware infected individuals who account for as many as 70% of new infections. One of the main challenges of current serology is detecting those who are infected but do not have measurable amounts of antibodies yet as they are in the Window Period (a stage referred to as acute HIV). Persons with acute HIV infection are 26 times more likely to transmit HIV than individuals with established infection. In addition, it has been found that Initiating treatment during the acute phase offers substantial benefits with every HIV infection averted saving over $600,000 in lifetime medical costs. The SMARTube can be integrated into routine HIV testing especially in high risk populations where detecting those acutely infected may have a significant impact in halting the epidemic.

In case of an HIV pregnant woman, detecting HIV early enough in the pregnancy can enable providing proper treatment to the mother and reducing the transmission to the fetus from 30% to 1-2% which would in fact mean saving 28-29 out of every 30 babies. As mentioned previously, there are a total of 130 million annual pregnancies worldwide with many countries implementing routine HIV screening as part of all pregnancies. In addition, the SMARTube can be used as part of testing of healthcare workers experiencing a needle stick injury (over 37 million global incidents annually) as well as testing of additional high risk populations such as prisoners (approx. 10 million globally) and security forces (military, police, etc').

SMARTube™
The SMARTube™ is a blood sample processing devices for improving specificity and sensitivity of serological assays for infections such as: HCV, HIV, HBV, XMRV, CMV, HTLV-1, Chagas, Tuberculosis and others, a market of hundreds of millions of tests a year.

The SMARTube is a device that is essentially a tissue culture tube with a proprietary solution. This device facilitates a small blood sample to be incubated prior to testing for the presence of antibodies using commercially available assays. SMARTube enables super-stimulation of primed lymphocytes, leading to enhanced production of HIV antibodies in vitro. It is a complementary product for improving performance of standard serological assays by reducing the rate of both false positive and false negative results. SMARTube enables detection of infections during the Window Period (when the standard serology is negative), enables validation of a positive infection and also effectively distinguishes between various stages of infection: acute, chronic, active or viral cleared/resolved infection (be it spontaneous or drug elicited), while the standard serological test cannot produce this information.

One mL of whole blood, collected in commercially available heparin or EDTA blood collection tubes, is introduced into the SMARTube (via sterile pipette or equivalent means). The SMARTube containing the blood sample is then incubated at 37°C in a humidified 5% CO2 incubator for three to five days. After incubation, an aliquot of the supernatant fluid "SMARTplasma", (which separates from the rest of the blood elements by gravity during the incubation period), is removed for testing using a commercially available standard ELISA test or any other method suitable for the detection and/or quantification of antibodies.

SMARTube has developed three products: SMARTube™HCV, SMARTube™HIV and combined SMARTube™HIV&HCV. Each product includes several models depending on the form of blood draw (direct vs. indirect), the anticoagulants used (Heparin, EDTA, Citrate) and the form of incubation (CO2, non-CO2).

Since most of the serological testing is being done via automated systems, it is imperative that any product introduced into such settings has the ability to integrate into the existing set-up. The SMARTube has been developed so it can integrate into the lab routine. Once 1 ml of whole blood is transferred into the SMARTube, it is placed in an incubator following which it can be placed in the automated testing system just like a regular blood collection tube. The automated system then draws the SMARTplasma sample from the SMARTube as it would normally do.

The Stimmulation Index (SI) using the SMARTube
As mentioned previously, current serology does not enable distinction between various stages of an infection (active, resolved, acute, chronic, primary, secondary, etc'). The Stimmunology Index (SI) was developed for enabling such a distinction using current serology. In this approach, the levels of antibodies are measured in both plasma and SMARTplasma. The figure below is a graph depicting the expected change in antibody levels in both types of samples over time during the course of infection. As can be seen, the reduction in antibody levels in SMARTplasma happens shortly after clearance of an infection. This is due to the fact that the levels of antibodies in the SMARTplasma is dependent on both the antibodies already present in the blood, and newly produced antibodies, in culture, by virus primed B-cells. With primed cells gone from the blood shortly after the clearance of the virus from the circulation, in vitro production ceases too. Thus, the decrease of antibody levels in SMARTplasma precedes the one observed in the plasma. However, if the infection is chronic, there is a small set of newly primed B-cells in the blood, which maintains antibody levels in plasma and, following stimulation in culture, produces antibody into SMARTplasma. Based on these two different patterns, the ratio between the antibody levels in the SMARTplasma and the antibody level in the plasma, termed Stimmulation Index (SI), differs according to the stage of infection. As can be seen, Confidential 13 there are four different possible SI values, and each one could serve as an indicator of a different stage in the infection:
• SI=∞ indicates very early infection, when antibodies in the SMARTplasma appear prior to seroconversion. Dividing a positive reading of the SMARTplasma by zero, as the plasma in negative for antibodies, yields an infinitesimal (∞) value.
• SI>>1 indicates recent seroconversion due to infection. The antibody production in vivo has not yet reached its full capacity, and thus it can be further stimulated in vitro. This leads to higher levels of antibodies in SMARTplasma than in concomitant plasma.
• SI~=1 indicates long term infection which has not cleared. The antibody production in vivo is at its full capacity, and no further stimulation is detected in the SMARTplasma.
• SI<<1 indicates cleared infection. Although there are still high levels of antibodies in the plasma, there is no further priming of B-cells, and thus no production of antibodies in vitro. This leads to a decrease in antibody levels in SMARTplasma following the incubation, when compared to the level in the concomitant plasma.