1

CONSULTORES LTD No.55 Ivan Susanin Str., ap. 7 E-mail: office@prevodi.co Web Page: www. prevodi.co Tel: +359(0)899 45 00 31

КОНСУЛТОРЕС ООД ул. Иван Сусанин No. 55, ап. 7

E-mail: office@ prevodi.co Web Page: www. prevodi.co

Тел: +359(0)899 45 00 31

Translation from Bulgarian

To: The Manager of "Immunotech Laboratories BG" Ltd. Mr. Dimitar Savov

FINAL REPORT

Regarding

SINGLE-CENTER PARALLEL-GROUP TRIAL WITH AN OPEN-LABEL AND TWO TREATMENT ARMS,

TO EVALUATE THE EFFICACY AND SAFETY OF 16-WEEKS OF INJECTION ADMINISTRATION OF ITV-1-

IMMUNH IN COMBINATION WITH AN ANTIRETROVIRAL THERAPY IN HIV-POSITIVE PATIENTS WITH

ADVANCED AIDS”

Protocol code: 9581958

Final version: 2.0

Dated: 07 August, 2014

Chief Researcher Assoc. Prof. Dr. Ivaylo Elenkov Summary of the clinical trial protocol: Name of the researched medical product: ITV-1-lmmunH (Immunotherapeutic vaccine) Research Center: Department for treatment of acquired immune deficiency at the Specialized Hospital for Active Treatment of Infectious And Parasite Diseases – “Prof. Ivan Kirov”, Sofia

Regarding:

HIV-positive patients with advanced stage (II, III, IV) of AIDS

Design of the trial:

Single-center, an open-label, parallel-group clinical trial with two treatment arms;

Clinical phase: "I";

DamienText Box

Tasks:

The present clinical trial aims to investigate the safety and efficacy of the parallel administration of ITV-

1-lmmunH with antiretroviral medicines compared to the independent administration of antiretroviral medicines in HIV-positive patients in an advanced stage of AIDS.

For the determination of the indicators of efficacy and safety a comparative analysis shall be conducted

between the clinical and laboratory parameters of both groups of patients participating in the clinical

trial: those, receiving combined treatment with ITV-1-lmmunH and antiretroviral therapy and those,

receiving treatment with antiretroviral therapy only.

Duration of the treatment:

Two courses of 8 weeks each with a 12-day break after the completion of the first course

Investigational medication:

An active component with molecular weight of 36 KD and 96% purity: sterile suspension of 12 mg of ITV-

1-lmmun, where each milliliter of injectable suspension contains 4 milligrams of active substance.

Reference treatment:

Antiretroviral therapy (ART) in a stable dose prior to the test

Dosage and administration:

IMP is administered by intramuscular injection two consecutive days, 3 ml within each week.

Number of patients: A minimum of 30 patients will be included in each treatment arm, and the total number will not exceed 100 patients.

Including criteria:

• Men and women at age from 18 to 60 years, who have voluntarily signed and dated an informed consent to participate in the trial under the applicable laws.

• With respect to women at childbearing age: negative blood pregnancy test at the screening visit who use and have given their consent to continue using the following methods to prevent pregnancy during and for at least 30 days after completion of the trial: oral contraceptives taken in a stable dosage at least one month before the screening visit; contraceptive implants or injectable contraceptives administered at least one month before the screening visit; intrauterine device; barrier methods plus spermicidal gel; sterile partner; abstaining from intercourse. The women who take oral contraceptives must further use barrier methods of contraception plus spermicidal gel or to abstain from intercourse during the trial.

• Infected with HIV - positive ELISA test within 12 months before the screening visit. • Number of CD4 + below 800 cells per cubic millimeter (mm3) established during the screening

visit

• Viral load above 5000 copies of HIV-1 RNA per milliliter established by polymerase chain reaction during the 14 days prior to the screening visit

• Patients at second, third or fourth stage of the disease. Treatment with antiretroviral chemotherapeutic agents

Excluding criteria:

1. Under the age of 18 years 2. Pregnant women and breastfeeding mothers 3. Patients with known allergy to any of the components of the investigational medication 4. A history of myocardial infarction or ischemic stroke 12 months before the screening visit. 5. Diseases of the gastrointestinal tract - stomach ulcer or duodenal ulcer, chronic

gastritis and colitis. 6. Patients with congenital or acquired immune defect or immunosuppression due to

medications. 7. Patients with hemophilia or other contraindications to administration of intramuscular

injections. 8. Patients treated or who have been treated with cytotoxic therapy with regard to a malignancy

within 30 days prior to the first dosage of the investigational medication. 9. Patients with count of CD4 + below 200 per cubic millimeter determined by flow cytometric

analysis. 10. Clinically significant changes in laboratory values suggesting the presence of significant or

non-controlled disease, which, according to the opinion of the researcher, may affect the interpretation of the results of the clinical trial.

11. Patients who have been treated or are treated within 60 days prior to the screening visit with the following drugs: immunomodulatory therapy, in particular IL-2, granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF) , intravenous administration of immunoglobulin, cytokines, plasmapheresis, steroid antirheumatic components, leukotriene antagonists, cyclosporine, methotrexate, interferon alpha, erythropoietin, thalidomide or agents stimulating the activity of macrophages.

12. Patients with a positive test for surface antigen of hepatitis B and/or positive antibodies for Hepatitis C established during the screening visit.

13. Patients with values of alanine aminotransferase (ALT), aspartate aminotransferase (ALT) and serum bilirubin more than three times above the upper reference limit of the norm for the respective test.

14. Participation in another clinical trial for testing a medicinal product 4 (four) weeks before the screening visit for this clinical trial.

15. History of substance abuse. 16. Lack of desire or opportunity to participate in the trial and to attend the research center in

accordance with the plan for the trial. 17. Patients who are unable to make their own decision to participate in the clinical trial.

Plan for the clinical trial:

The patients in the experimental group will conduct a total of 32 visits to the center for the clinical trial, and each therapeutic course will include 16 patient visits, with a break of 12 days between the two courses. The patients in the control group will conduct three visits to the center for the clinical trial. During the Visit Day 1 (screening), Visit Day 63 (end of the 12-day break) and Visit Day 126 (after

the second course of treatment) the patients in both groups (experimental and control) will be tested in order to evaluate the efficacy and safety of the administered treatment. During the other visits – from Visit Day 2 to Visit Day 51 during the first course of the treatment and from Day Visit 64 to Visit Day 114 during the second course of treatment, the patients in the experimental group will visit the research center for the administration of the investigational medication. Efficacy indicators:

Primary efficacy indicators:

• The proportion of patients who have achieved therapeutic success, manifested in: stabilization or improvement, resulting in an increase in the number of the following lymphocyte populations: CD3, CD16, CD56, CD45, CD4, CD19 and the correlation CD4 / CD8. • Stabilization or improvement, resulting in a decrease in the following lymphocyte

population: CD8 • Reduction of the viral load (measured as copies of RNA in 1 ml blood serum) with polymerase

chain reaction. Secondary efficacy indicators:

• The proportion of patients who have achieved therapeutic success, manifested in: Stabilization with regard to the general condition and reduction in the intensity or failure of some of the following clinical signs: fever, headache, fatigue, lack of appetite, pain at the injection spot, nausea, vomiting, diarrhea, yellowing of the skin, skin rashes.

• The proportion of patients who have achieved therapeutic success, manifested in: • stabilization or increase in the body weight and the body mass index (BMI).

Safety indicators:

The safety analysis of patients during the clinical trial will be based on monitoring of the following

parameters:

• experienced adverse events during the trial;

• clinically significant changes occurring in the vital indicators;

• Clinically significant changes occurring in the haematological and biochemical parameters

during the clinical trial.

Principles of the statistical analysis:

With respect to the primary efficacy indicators, a logistic regression analysis will be used to compare the proportion of patients who have achieved therapeutic success in both the experimental and the control group during the administration of the independent and combined analysis. With regard to the analysis of the primary efficacy indicators, Type I error 0.001 will be used for intermediate evaluation which allows for Type I error of 0.049 using bilateral tests, which will be used for the final analysis.

Scheduled commencement date of the trial: October 2015

Scheduled completion date of the trial: August 2016

Clinical Trial Design

The clinical trial will make comparative assessment of the efficacy and safety related to the

administration of ITV-1-lmmunH in patients with diagnosed HIV infection while applying antiretroviral

therapy. For that purpose, HIV-positive patients in an advanced stage of AIDS, who have voluntarily

signed an informed consent to participate in the clinical trial, will be divided randomly into two groups -

experimental and control. The participants in the experimental group will receive the investigational

product - ITV-1-lmmunH in the form of intramuscular injections within 2 successive treatment courses

with a 10-day break between them while receiving antiretroviral therapy. The participants in the control

group will be monitored while receiving antiretroviral therapy. Within the 2 groups the participants will

be further stratified according to the number of CD4 + T- lymphocytes. The investigational medication

(ITV-1-lmmunH) will be administered to the patients by intramuscular injections in two consecutive days

during each week. The total treatment period will be 16 weeks with a 10-day break between the two

treatment courses.

The clinical testing schedule includes a screening visit, during which all the patients, who pass the

screening and have no contraindications for participation in the trial, will be administered the first

dosage of the investigational medication. After that, 15 more visits will be conducted (from Day 2 to Day

51), during which the patients will visit the research center within 2 consecutive days each week in order

to receive injections with the investigational medication and will be monitored for adverse events. After

the visit, on Day 51, a 12-day break will take place, at the end of which Visit Day 63 will be held in order

to assess the condition of the patients and to take blood samples to determine the indicators of efficacy

and safety. Later, the patient will receive the second cycle with injections with the investigational

medication by means of 16 visits to the research center (from Day 64 to Day 114) and on Day 126 the

final visit will be held. The total duration of the participation of the patients in the clinical trial will not

exceed 18 weeks and this period includes as well the 12-day break between the two courses of ITV-1-

lmmunH. Upon completion of their participation in the trial, the patients will continue their treatment

with antiretroviral medications and visits to the research center for disease control.

The present clinical trial will be conducted in accordance with the Declaration of Helsinki (1964 and the

subsequent supplements), the current rules of the Good Clinical Practice and the applicable European

and local laws. After the completion of the trial, the patients will be given the right to continue their

treatment in accordance with the good clinical practice and at the discretion of their physician.

Criteria for withdrawal of patients from the clinical trial.

The patients have the right to withdraw from the trial at any time and at their own discretion, without

affecting the attitude towards them by the researcher and their subsequent medical treatment. The

administrated medication may be terminated or suspended by the contracting authority at any time of

the trial because of one (or more) of the following reasons:

1. At the sole discretion of the patient or the discretion of the researcher;

2. The occurrence of an excluding criterion during the clinical trial;

3. Failure to observe the respective procedures by a patient or unwillingness to comply with the

procedures for participation in the clinical trial, which would suggest an increase in the risk for his/her

health during the trial.

4. Significant deviation from the clinical trial procedures;

5. Need to administrate another treatment that has not been approved in the present Protocol;

6. Withdrawal in the interest of the patient at the discretion of the researcher ;

7. Withdrawal of the signed by the patient informed consent;

8. Termination of the clinical trial by the contracting authority;

9. Pregnancy during the participation in the clinical trial.

A follow-up visit, scheduled for Day 126 (end of the trial), will be conducted with regard to all the

patients who withdraw from the clinical trial before the end of the treatment. The visit will be held

on the day of withdrawal of the patient from the trial.

Information on the administrated treatment.

The contracting authority with regard to the clinical trial will be obliged to deliver the

investigational medicinal product (IMP) to the center for the conducting of the clinical trial,

providing to the researcher or to the appointed pharmacist (if applicable) all the necessary

analytical certificates for the production of the IMP in accordance with the requirements of the

Good Manufacturing Practice (GMP) and the local legislation.

The researcher (or the appointed pharmacist, if applicable) is responsible for the proper storage of all

medications given to the patients during the clinical trial in a safe and secure location with limited

access of people and observing the required temperature and humidity (if applicable) of storage.

Patient samples

The trial includes a total of 66 patients separated into two groups - main and control. 35 people were

initially included in the basic group, three of whom did not complete the trial due to physical

impossibility to further participate, and one of the patients was excluded due to an allergic reaction

during the initial dermatological test for tolerance.

Thus, the clinical trial was completed by 31 patients in each of the groups.

The basic group contained 26 men and 5 women eligible for inclusion; all of them were between 28

and 60 years old, average age - 44 years. All the women met the criteria for inclusion after the

presentation of a medical certificate for absence of pregnancy, according to the trial protocol.

Depending on the degree of immunodeficiency the patients in the main group were divided into

three sub - groups - with CD4

400 - 18 people.

The participants in both groups of the clinical trial received antiretroviral therapy (ART), which

included combined treatment regimens, based on 2 nucleoside reverse transcriptase inhibitors and,

respectively, non-nucleoside reverse transcriptase inhibitor or boosted with ritonavir protease

inhibitor.

Trial Results

FINAL REPORT- ANALYSIS of the conducted immunological tests of blood samples under contract №

214/ 19.10.2015 between "Immunotech Laboratories BG" Ltd. and the National Center for Infectious

and Parasitic Diseases (NCIPD), Sofia

Pursuant to eth contract № 214/19.10.2015 between "Immunotech Laboratories BG" Ltd. and NCIPD,

Sofia, related to the clinical trial with a protocol code - 14031958, with contracting authority

(sponsor) - Immunotech Laboratories BG - Eudra CT 2014-003465-26, during the period 02.11.2015-

01.06.2016 at NRL of Immunology immunological coded blood tests were conducted with regard to

patients with HIV-1 infection, selected and sent by the Department for treatment of acquired

immune deficiency at the Specialized Hospital for Active Treatment of Infectious And Parasite

Diseases – “Prof. Ivan Kirov”, Sofia,. For the period 02.11.2015 – 14.05.2016, a total of 115

immunological tests were conducted, taking into consideration the indicators provided for in the

contract, as follows: absolute number (AN) of the general lymphocytes, percentage and absolute

number of T (CD3 +) lymphocytes, B (CD19 +) lymphocytes and NK ( CD56 + 16 +) cells, percentage

and absolute number of T-lymphocyte subpopulations CD4 T (CD4 + CD3 +), CD8 T (CD8 + CD3 +), and

their ratio (CD4/CD8).

31 patients had a full course of follow-up (3-times). Nine have given up after the first test; 5 (five)

patients had two tests each. Three (3) tests outside the schedule were made. The results from the

first, second and third tests were summarized for those patients with a full course of follow-up. The

results from all the tests have been applied in a table (Appendix 1) and have been presented to the

main researcher as individual trial protocols.

Results from the baseline measurements

The average (min-max) values of the studied populations are summarized in Table 1. The absolute

number of CD4 T lymphocytes is (average, min. - max.) 528 (116 - 1.261) cells/mcl and the

percentage: 22 (4- 44)%. The distribution of patients according to degree of immunodeficiency during

the first measurements is as follows: 9% (n = 3) with severe immunodeficiency (CD4 AC 400). Respectively, in the majority of the patients (81%, n =

25), the CD8 T cells are relatively increased, and the CD4/CD8 index - reduced below 0.9 (93.5%, n =

29).

The rest of the indicators: lymphocyte count (average, min. - max.) 2.379 (0.604 - 4.835) with

deviations from the norm in 22.5% (7 people): 2 of them with reduced lymphocyte count (3100);

In the cells (average, min. - max.) 0.221 (0.044 to 0.425) with deviations from the norm in 71%,

respectively: absolute decrease in 52% (n = 16), relative decrease - in 19%, (n = 6) ; NK cells (average,

min. - max.) 0.341 (0.07 - 0.846) with deviations from the absolute number in 10% (n = 3),

respectively - below the lower reference limit in two patients and above the upper reference limit - in

one patient; the relative share is decreased in one patient and it is above the upper limit in two

patients.

Results from the second measurements

The results are summarized in Table 1. We notice an increase in the absolute number and the relative

share of: B cells (Wilcoxon's p

Appendix 1: Results from all the immunological tests pursuant to the contract N° 214/ 19.10.2015 Total number of

tests Code Date of the

analysis Lymphocytes General

Т cells CD3+

Т-helper indus CD3+CD4

+

Cytotoxic

Т cells

CD3+CD8

+

General В

cells CD19+

NK cell

CD3- CD56+

General Т

cells CD3+

Т-helper indus CD3+CD

4+

CytotoxicТ

cells

CD3+CD8+

General В

cells CD19+

NK cells

general CD3-

CD56+

Index CD4/CD8

1 212 08.10.2015 1,796 1,404 0,298 1,062 0,140 0,208 78,2 16,6 59,1 7,8 11,6 0,28

2 212 10.12.2015 1,684 1,169 0,302 0,815 0,237 0,262 69,4 17,9 48,39 14,05 15,6 0,37

3 212 11.02.2016 2,028 1,346 0,367 0,897 0,282 0,375 66,4 18,1 44,2 13,9 18,5 0,41

4 525 08.01.2016 2,123 0,965 0,340 0,507 0,425 0,726 45,5 16,0 23,88 20,00 34,2 0,67

5 525 08.03.2016 2,366 1,244 0,481 0,621 0,475 0,634 52,6 20,3 26,25 20,08 26,8 0,77

6 525 10.05.2016 2,354 1,181 0,452 0,596 0,426 0,729 50,2 19,2 25,33 18,11 31,0 0,76

7 900 08.10.2015 2,723 2,128 0,260 1,726 0,349 0,193 78,2 9,6 63,4 12,8 7,1 0,15

8 900 14.12.2015 3,205 2,434 0,337 1,972 0,486 0,281 76,0 10,5 61,5 15,2 8,8 0,17

9 900 17.02.2016 2,867 2,159 0,311 1,716 0,395 0,304 75,3 10,9 59,8 13,8 10,6 0,18

10 1409 05.11.2015 2,414 1,928 0,629 1,155 0,135 0,342 79,9 26,1 47,8 5,6 14,2 0,54

11 1409 04.01.2016 2,850 2,303 0,742 1,374 0,168 0,368 80,8 26,0 48,2 5,9 12,9 0,54

12 1409 09.03.2016 4,189 3,163 1,112 1,718 0,489 0,503 75,5 26,6 41,0 11,7 12,0 0,65

13 1473 07.12.2015 1,459 1,108 0,418 0,674 0,108 0,240 76,0 28,7 46,2 7,37 16,5 0,62

14 1473 05.02.2016 1,355 1,028 0,406 0,599 0,098 0,224 75,8 30,0 44,2 7,23 16,5 0,68

15 1473 14.04.2016 1,862 1,421 0,556 0,838 0,116 0,310 76,3 29,9 45,0 6,23 16,7 0,66

16 1550 22.12.2015 2,823 2,445 0,773 1,522 0,148 0,224 86,6 27,39 53,9 5,26 7,9 0,51

17 1550 23.02.2016 3,054 2,680 0,880 1,645 0,116 0,243 87,8 28,83 53,8 3,79 7,9 0,54

18 1550 25.04.2016 3,463 3,020 0,989 1,796 0,157 0,279 87,2 28,56 51,9 4,54 8,1 0,55

19 1640 07.01.2016 1,582 1,018 0,287 0,687 0,201 0,351 64,4 18,1 43,4 12,71 22,2 0,42

20 1640 09.03.2016 1,855 1,266 0,383 0,812 0,242 0,330 68,3 20,7 43,8 13,06 17,8 0,47

21 1640 10.05.2016 1,694 1,094 0,350 0,677 0,211 0,373 64,6 20,7 40,0 12,46 22,0 0,52

22 1643 23.12.2015 1,319 0,807 0,407 0,358 0,209 0,301 61,2 30,8 27,1 15,8 22,8 1,14

23 1760 29.12.2015 1,934 0,813 0,315 0,496 0,212 0,846 42,0 16,0 26,0 11,0 44,0 0,64

24 1760 29.02.2016 1,421 0,631 0,271 0,350 0,202 0,459 44,4 19,1 24,6 14,2 32,3 0,78

25 1760 03.05.2016 1,911 0,761 0,318 0,425 0,244 0,876 39,8 16,6 22,3 12,8 45,8 0,75

26 1790 16.11.2015 0,971 0,790 0,240 0,530 0,097 0,081 81,4 24,8 54,6 10,0 8,3 0,45

27 1790 25.01.2016 1,804 1,426 0,421 0,954 0,169 0,207 79,0 23,3 52,9 9,4 11,5 0,44

28 1790 08.04.2016 1,300 0,999 0,335 0,627 0,169 0,129 76,8 25,7 48,3 13,0 9,9 0,53

29 1814 02.11.2015 3,712 2,808 0,845 1,898 0,420 0,482 75,7 22,8 51,1 11,3 13,0 0,44

30 1814 04.01.2016 4,038 3,093 0,860 2,158 0,386 0,553 76,6 21,3 53,4 9,5 13,7 0,40

31 1814 07.03.2016 3,892 3,040 0,952 1,978 0,355 0,413 78,1 24,5 50,8 9,1 10,6 0,48

32 1830 10.12.2015 3,026 2,387 0,690 1,476 0,134 0,494 78,9 22,8 48,79 4,42 16,3 0,47

33 1834 08.10.2015 1,373 0,983 0,302 0,643 0,275 0,095 71,6 22,0 46,9 20,0 6,9 0,47

34 1834 09.12.2015 2,155 1,494 0,450 0,973 0,406 0,244 69,3 20,9 45,2 18,83 11,3 0,46

35 1834 17.02.2016 1,523 1,084 0,396 0,636 0,311 0,118 71,2 26,0 41,8 20,44 7,8 0,62

36 1837 12.10.2015 2,327 1,903 0,421 1,303 0,251 0,151 81,8 18,1 56,0 10,8 6,5 0,32

37 1837 16.12.2015 2,514 2,004 0,538 1,300 0,322 0,185 79,7 21,4 51,7 12,8 7,3 0,41

38 1837 15.02.2016 2,965 2,365 0,602 1,601 0,350 0,222 79,8 20,3 54,0 11,8 7,5 0,38

39 1851 09.11.2015 1,862 1,392 0,580 0,811 0,170 0,293 74,8 31,1 43,5 9,1 15,7 0,72

40 1851 11.01.2016 1,880 1,545 0,697 0,828 0,146 0,186 82,2 37,1 44,1 7,7 9,9 0,84

41 1851 15.03.2016 1,638 1,285 0,577 0,656 0,131 0,202 78,5 35,2 40,0 8,0 12,3 0,88

42 1851 04.12.2015 1,300 0,992 0,420 0,578 0,114 0,189 76,3 32,3 44,5 8,7 14,6 0,73

43 1853 16.10.2015 2,341 1,798 0,597 1,170 0,162 0,374 76,8 25,5 50,0 6,9 16,0 0,51

44 1853 21.12.2015 2,192 1,686 0,595 1,055 0,158 0,342 76,9 27,2 48,1 7,2 15,6 0,56

45 1853 22.02.2016 1,855 1,420 0,568 0,814 0,166 0,258 76,6 30,6 43,9 8,9 13,9 0,70

46 1855 16.10.2015 4,233 3,102 0,767 2,162 0,561 0,552 73,3 18,1 51,1 13,3 13,0 0,35

47 1869 16.10.2015 1,952 1,175 0,471 0,683 0,307 0,412 60,2 24,1 35,0 15,7 21,1 0,69

48 1869 22.12.2015 2,397 1,497 0,607 0,852 0,395 0,492 62,4 25,3 35,5 16,5 20,5 0,71

49 1869 29.02.2016 2,148 1,392 0,582 0,776 0,404 0,259 64,8 27,1 36,1 18,8 12,1 0,75

50 1870 16.10.2015 2,074 1,601 0,514 1,021 0,296 0,165 77,2 24,8 49,2 14,3 8,0 0,50

51 1870 22.12.2015 1,094 0,793 0,250 0,517 0,205 0,093 72,4 22,9 47,3 18,7 8,5 0,48

52 1870 29.02.2016 1,652 1,331 0,455 0,820 0,202 0,104 80,6 27,6 49,7 12,3 6,3 0,56

53 1874 17.12.2015 2,804 2,051 0,712 1,173 0,376 0,372 73,1 25,4 41,84 13,4 13,3 0,61

54 1874 16.02.2016 2,914 2,119 0,770 1,122 0,279 0,500 72,7 26,4 38,51 9,6 17,2 0,69

55 1874 03.05.2016 3,907 2,803 1,043 1,468 0,505 0,585 71,8 26,7 37,57 12,9 15,0 0,71

56 1878 06.11.2015 2,709 2,046 0,505 1,312 0,198 0,462 75,5 18,7 48,5 7,3 17,1 0,39

57 1878 12.01.2016 4,075 3,056 0,756 1,997 0,413 0,596 75,0 18,5 49,0 10,13 14,6 0,38

58 1878 14.03.2016 2,842 2,022 0,506 1,184 0,264 0,544 71,1 17,8 41,6 9,28 19,1 0,43

59 1900 22.12.2015 4,793 3,950 0,997 2,692 0,364 0,477 82,4 20,81 56,2 7,59 9,9 0,37

60 1900 26.02.2016 2,795 2,280 0,854 1,225 0,293 0,212 81,6 30,5 43,81 10,5 7,6 0,70

61 1900 25.05.2016 3,598 2,941 1,073 1,647 0,378 0,269 81,7 29,8 45,77 10,5 7,5 0,65

62 1910 16.10.2015 2,715 2,298 0,659 1,499 0,152 0,259 84,7 24,3 55,2 5,6 9,5 0,44

63 1959 04.11.2015 1,875 1,145 0,295 0,820 0,177 0,533 61,1 15,7 43,7 9,5 28,4 0,36

64 1959 11.01.2016 2,358 1,503 0,413 1,048 0,262 0,580 63,7 17,5 44,4 11,1 24,6 0,39

65 1959 21.03.2016 2,259 1,343 0,361 0,930 0,181 0,703 59,5 16,0 41,2 8,0 31,1 0,39

66 1971 26.11.2015 1,913 1,435 0,403 0,927 0,150 0,321 75,0 21,1 48,4 7,8 16,8 0,43

67 1983 26.10.2015 1,808 1,357 0,650 0,681 0,127 0,320 75,0 36,0 37,7 7,0 17,7 0,96

68 1983 05.01.2016 2,255 1,697 0,835 0,826 0,240 0,316 75,2 37,0 36,6 10,6 14,0 1,01

69 1983 07.03.2016 2,199 1,701 0,836 0,792 0,221 0,255 77,3 38,0 36,0 10,1 11,6 1,06

70 2003 03.11.2015 1,829 1,382 0,411 0,878 0,308 0,128 75,6 22,5 48,0 16,8 7,0 0,47

71 2003 05.01.2016 1,978 1,496 0,445 0,959 0,300 0,171 75,6 22,5 48,5 15,2 8,7 0,46

72 2003 08.03.2016 2,379 1,850 0,538 1,173 0,313 0,195 77,8 22,6 49,3 13,2 8,2 0,46

73 2004 11.11.2015 3,192 2,259 0,731 1,470 0,459 0,466 70,8 22,9 46,1 14,4 14,6 0,50

74 2004 28.01.2016 3,314 2,417 0,971 1,383 0,447 0,442 72,9 29,3 41,7 13,5 13,3 0,70

75 2014 02.12.2015 0,646 0,389 0,116 0,265 0,189 0,061 60,1 17,9 41,0 29,3 9,5 0,44

76 2015 17.11.2015 3,729 2,843 0,567 2,255 0,381 0,497 76,2 15,2 60,5 10,2 13,3 0,25

77 2015 19.01.2016 2,927 2,138 0,571 1,550 0,364 0,423 73,0 19,5 52,9 12,4 14,5 0,37

78 2015 24.03.2016 2,948 2,251 0,502 1,727 0,287 0,399 76,4 17,0 58,6 9,7 13,5 0,29

79 2025 20.11.2015 2,518 1,760 0,139 1,578 0,348 0,405 69,9 5,5 62,7 13,8 16,1 0,09

80 2049 07.01.2016 4,835 4,276 1,261 2,818 0,161 0,393 88,4 26,1 58,3 3,33 8,1 0,45

81 2049 07.03.2016 3,259 2,845 0,966 1,716 0,142 0,263 87,3 29,6 52,6 4,35 8,1 0,56

82 2049 09.05.2016 4,009 3,496 1,232 2,141 0,160 0,336 87,2 30,7 53,4 4,00 8,4 0,58

83 2050 02.11.2015 3,150 2,326 0,123 2,138 0,125 0,696 73,8 3,9 67,9 4,0 22,1 0,06

84 2050 05.01.2016 2,711 2,021 0,166 1,803 0,155 0,532 74,6 6,1 66,5 5,7 19,6 0,09

85 2050 09.03.2016 2,449 1,835 0,184 1,561 0,107 0,496 74,9 7,5 63,7 4,4 20,3 0,12

86 2050 02.12.2015 2,287 1,756 0,177 1,540 0,146 0,375 76,8 7,7 67,4 6,4 16,4 0,11

87 2053 03.11.2015 3,326 2,399 0,380 1,755 0,506 0,404 72,1 11,4 52,8 15,2 12,1 0,22

88 2053 05.01.2016 3,135 2,285 0,380 1,665 0,383 0,461 72,9 12,1 53,1 12,2 14,7 0,23

89 2063 23.11.2015 2,272 1,820 0,116 1,539 0,123 0,324 80,1 5,1 67,7 5,4 14,3 0,08

90 2063 20.01.2016 2,229 1,774 0,122 1,525 0,160 0,290 79,6 5,5 68,4 7,2 13,0 0,08

91 2063 25.03.2016 3,009 2,323 0,160 1,876 0,232 0,411 77,2 5,3 62,4 7,7 13,6 0,09

92 2072 20.11.2015 3,755 3,340 0,354 2,804 0,264 0,141 88,9 9,4 74,7 7,0 3,8 0,13

93 2072 19.01.2016 3,764 3,273 0,394 2,708 0,296 0,180 86,9 10,5 71,9 7,9 4,8 0,15

94 2072 07.04.2016 4,081 3,638 0,515 2,926 0,280 0,154 89,1 12,6 71,7 6,9 3,8 0,18

95 2082 08.10.2015 2,056 1,618 0,170 1,446 0,051 0,354 78,7 8,3 70,4 2,5 17,2 0,12

96 2082 04.01.2016 2,476 2,061 0,251 1,759 0,069 0,342 83,2 10,1 71,1 2,8 13,8 0,14

97 2082 07.03.2016 2,250 1,902 0,232 1,609 0,073 0,263 84,5 10,3 71,5 3,3 11,7 0,14

98 2083 15.10.2015 0,604 0,477 0,258 0,209 0,044 0,070 79,0 42,7 34,7 7,3 11,6 1,23

99 2083 15.12.2015 1,509 1,194 0,573 0,566 0,203 0,106 79,1 38,0 37,5 13,5 7,0 1,01

100 2083 17.02.2016 1,343 1,069 0,595 0,445 0,188 0,064 79,6 44,3 33,1 14,0 4,8 1,34

101 2088 05.11.2015 1,740 1,276 0,420 0,778 0,281 0,175 73,3 24,1 44,7 16,1 10,0 0,54

102 2088 11.01.2016 2,117 1,615 0,588 0,925 0,270 0,225 76,3 27,8 43,7 12,8 10,6 0,64

103 2088 07.03.2016 1,777 1,326 0,507 0,717 0,276 0,164 74,6 28,6 40,3 15,5 9,2 0,71

104 2101 04.01.2016 1,967 1,484 0,589 0,724 0,267 0,211 75,4 29,9 36,8 13,6 10,7 0,81

105 2101 09.05.2016 2,709 1,962 0,872 0,843 0,341 0,384 72,4 32,2 31,1 12,6 14,2 1,03

106 2114 08.10.2015 1,071 0,855 0,242 0,580 0,044 0,135 79,8 22,6 54,2 4,1 12,6 0,42

107 2122 09.11.2015 2,316 1,687 0,540 0,959 0,180 0,447 72,8 23,3 41,4 7,8 19,3 0,56

108 2122 20.01.2016 2,500 1,634 0,510 0,958 0,217 0,642 65,4 20,4 38,3 8,7 25,7 0,53

109 2122 24.03.2016 2,961 2,066 0,794 0,976 0,268 0,615 69,8 26,8 32,9 9,0 20,8 0,81

110 2153 27.01.2016 1,487 1,126 0,384 0,633 0,151 0,207 75,7 25,82 42,55 10,14 13,9 0,61

111 2153 04.04.2016 2,004 1,492 0,430 0,852 0,198 0,292 74,5 21,48 42,51 9,89 14,6 0,51

112 2164 03.11.2015 1,573 1,008 0,357 0,557 0,159 0,393 64,1 22,7 35,4 10,1 25,0 0,64

113 2164 15.01.2016 1,515 0,980 0,434 0,472 0,178 0,349 64,7 28,6 31,2 11,78 23,0 0,92

114 2164 18.12.2015 1,065 0,724 0,341 0,333 0,156 0,181 67,9 32,0 31,3 14,7 17,0 1,02

115 2170 03.11.2015 2,930 2,772 0,517 2,162 0,069 0,082 94,6 17,6 73,8 2,3 2,8 0,24

FINAL REPORT - ANALYSIS of the conducted virological tests of blood samples pursuant to contract №

214/19.10.2015 between "Immunotech Laboratories BG" Ltd. and NCIPD, Sofia.

In the NRL for HIV, viral load tests (VT) were carried out with regard to 101 coded blood samples from 36 patients with HIV-1 infection, selected and sent by the Department for treatment of acquired immune deficiency at the Specialized Hospital for Active Treatment of Infectious And Parasite Diseases – “Prof. Ivan Kirov”, Sofia, in connection with the clinical research with a protocol code - 14031958; contracting authority (sponsor) Imunotech Laboratories BG - Eudra CT 2014-003465-26, during the period 02.11.2015 - 01.06.2016.

The blood samples were sent and analyzed in three stages. For the first group, samples of 36 patients were sent, for the second - 34 patients, and for the third group in the NRL for HIV were obtained 31 samples. Thirty-one of the patients were three times tested for VL (Table 1).

The tests for VL were conducted with Real-Time PCR Test for in vitro quantitative analysis of HIV-1 with the automated system Abbott Molecular's m2000, approved by the US FDA, and CE, marked for in vitro diagnostics in Europe. The extraction of the viral nucleic acids was conducted with an automatic extractor with magnetic particles - Abbott Molecular m2000sp; the Real-time amplification and detection were performed with Abbott Molecular m2000rt. The detection limit of the test is within 40 (1,6 log copies/ml) to 10000000 (7,0 log copies/ml) copies/ml at extraction of 500 mcl of plasma sample. The target region of the viral genome is a fragment from the integrase of the gene pol of HIV.

The results of the VL, which are below the lower detection limit of the test (

1874 17 71400 0 0 0

1878 18 164000 0 0 0

1900 19 249000 0 0 0

1959 20 3595 0 0 0

2003 21 1106530 0 0 54

2015 22 566931 0 56 155

2049 23 89546 0 0 0

2050 24 385454 0 0 0

2063 25 150753 0 0 0

2072 26 11292 0 0 0

2082 27 128299 83 86 105

2083 28 143826 140 123 67

2088 29 72959 0 0 0

2122 30 149743 0 0 0

1983 31 18500 17204 12521 34868

Table 2. Results from the tests of VL of the participants in the trial with 2 tests

Code of the patient Patient Initial VL 1 tested copies of VL

II tested copies of VL

2053 1 226615 0 0

2101 2 165624 154 42

2153 3 224101 51 0

Table 3. Results from the tests of VL of the participants in the trial with 1 test.

In the re-examination within the period of the trial (02.11.2015 – 01.06.2016) we established a decrease in the VT in

most of the tested subjects 33 (80.5%). (Table 1). In 5 (12.1%) patients we found detectable amount of HIV RNA

during the second test with values of VL, respectively: 259, 51, 44, 56 and 86, and in 4 (9.8%) patients we found

detectable levels of VL, and during the third test - 87, 56, 54 and 155. These levels of VL are close to the detection

limit of the test, they are not considered blip and are not associated with a lasting treatment failure in patients with

HIV. For one of the patients - № 36, no clinically significant decrease of VT during this trial was observed and a

clinically significant increase in VL was established.

Code of the patient Patient Initial VL 1 tested copies of VL

1910 1 114000 101

1855 2 387000 47

Figure 1. Trend of VL during the first, second and third test of 30 patients during the trial.

I test copies VL II test copies VL III test copies VL

Figure 2. Trend of VT during the first, second and third test of the patient with code 1983.

I test copies VL II test copies VL III test copies VL

Statistical processing of the output data of the carried out immunological tests of blood samples of patients with

HIV-1 infection.

Two groups of patients participate in the analysis– a main group (experimental, in which the new

treatment is applied) and a control group, which is identical to the experimental in every aspect, except that it

does not include the experimental treatment. In the following graphics and tables the main group will be

indicated as Case, and the control group - as Control. 31 patients had a full course of follow-up in the main

group. The same number of patients (31) is presented in the control group. The results from all conducted

tests have been applied in a tabular form (Appendix 1). The examined indicators will be CD4, CD8, and their

ratio CD4/CD8.

I. Variation analysis of the indicators CD4, CD8, as well as their ratio CD4/CD8.

The analysis will be applied to characterize the distribution of these indicators. Since CD4, CD8 and CD4/D8 are

quantitative variables they will be presented through average (mean) values, standard deviation (Standard

Deviation), minimum and maximum.

• CD4 - Case/Control Group

Case Control

Standard Standard Total

Mean Minimum Maximum Deviation Total N Mean Minimum Maximum Deviation N

CD4_1 475 116 1261 254 31 373 109 773 197 31

CD4_3 581 160 1232 287 31 381 110 842 198 31

Table 1 shows the mean, minimum, maximum and standard deviation of CD4 from the first date (CD4_1) and

the last date of taking blood samples (CD4_3).

Upon initial CD4 blood sampling in the main group (Case) it varies from a minimum of 116 to a maximum of

1261, and the mean is 475. The results from the third blood sampling range from 160 to 1232, and the mean

is 581.

In the control group (Control), the results for the first date are within the interval of a minimum of 109 to a

maximum of 773, and the mean is 373. During the third date they are respectively from 110 to 842, with a

mean of 381.

Figure 1

Figure 1 graphically illustrates the change in the mean value of CD4 with regard to the main and control group

during the three dates of blood sampling.

• CD8 - Case/Control Group

Case Control

Standard Standard

Mean Minimum Maximum Deviation Total N Mean Minimum Maximum Deviation Total N

CD8_1 1258 209 2818 708 31 1044 135 2665 611 31

CD8_3 1219 425 2969 604 31 862 165 2366 534 31

Table 2 summarizes the results from the first (CD8_1) and the last date of blood sampling (CD8_1) for the CD8

indicator in the main and the control group.

In the main group CD8 during the first date of blood sampling, it varies from 209 to 2818, and the mean is

1258. During the third date the minimum is 425, the maximum is 2969, and the mean is 1219.

With respect to the control group, CD8 on the first date is from 135 to 2665, and the mean is 1044. During the

last date the minimum is 165, the maximum is 2366, and the mean is 862.

Figure 2 shows the change of the mean value of the CD8 indicator for both groups during the three dates

of blood sampling.

• CD4/CD8 - Case/Control

Table 3

Group

Case Control

Standard Standard

Mean Minimum Maximum Deviation Total N Mean Minimum Maximum Deviation Total N

CD4/CD8_1 0,46 0,06 1,23 0,25 31 0,49 0,09 2,20 0,45 31

CD4/CD8_3 0,56 0,09 1,34 0,28 31 0,60 0,09 2,24 0,48 31

Table 3 presents the summarized characteristics of the CD4/CD8 ratio during the first and last date of blood sampling

for both groups (main and control). In the main group CD4/CD8 during the first date the values are from 0.06 to 1.23,

and the mean is 0.46. During the third date the values are from 0.09 to 1.34 and the mean is 0.56.

During the first date for the control group CD4/CD8 varies from 0.09 to 2.2, and the mean is

0.49. During the third date the minimum is 0.09, the maximum is 2.24 and the mean is 0.6.

Figure 3 depicts the graphical mean value of the CD4/CD8 ratio in the two groups during the three dates of blood

sampling.

I. Verification of the hypothesis.

There is an increase in the mean values of CD4 from the first and the last date of blood sampling in both groups

(main and control). It is necessary to establish whether this increase is statistically significant. This will be done using

a scientific hypothesis.

The scientific hypothesis is an assumption which must be verified in an empirical way. For this purpose, the scientific

hypothesis is reformulated in one of the following two statistical hypotheses:

Null (working) hypothesis (H0) which claims that the parameters of the compared data do not differ (no significant

difference).

An alternative hypothesis (Hi), which claims that the difference, which is observed in the data is statistically

significant (essential).

The conclusions made have a probabilistic nature. The degree of certainty, with which the alternative hypothesis is

assumed to be true, is called guaranteed probability (P). The probability for the null hypothesis to be true, is called

significance level (a). The analysis will use the following limits for the guaranteed probability (P) and the significance

level (a):

P = 95%, to which corresponds a = 0.05

• CD4 – First and third date

In this case, the null hypothesis (H0) will state that there is no statistically significant increase in the average value of

CD4 between the first and the last date in both groups. The alternative hypothesis (H1) will state that the difference

observed is statistically significant.

Since the quantitative variable CD4 has a normal distribution, we will use a parametric criterion for the verification of

the hypotheses and in particular the Student’s t-criterion.

Table 4

Group Statistics

Group N Mean Std. Deviation Std. Error Mean

CD4_1 Case 31 475,26 254,164 45,649

Control 31 373,32 196,798 35,346

CD4 3 Case 31 580,65 287,187 51,580

Group N Mean Std. Deviation Std. Error Mean

CD4_1 Case 31 475,26 254,164 45,649

Control 31 373,32 196,798 35,346

CD4_3 Case 31 580,65 287,187 51,580

Control 31 381,03 198,258 35,608

Table 4 contains information about the distribution of the values in the compared groups - volumes (1\1), average

(Mean) values, standard deviation (Std. Deviation) and standard errors (Std. Error Mean). Table 5 Independent Samples Test

Levene's Test for

Equality of

Variances

t-test for Equality of Means

F Sig. t df

Sig.

(2-

tailed)

Mean

Difference Std. Error

Difference

95% Confidence Interval of the

Difference

Lower Upper

CD4_1 Equal variances assumed

Equal variances not

assumed

,692 ,409

1.766

1.766

60

56,461

,083

,083

101.935

101.935

57.734

57.734

-13,549

-13,698

217,420

217,569

CD4_3 Equal variances assumed

Equal variances not

assumed

1,969 ,166

3.185

3.185

60

53,302

,002

,002

199.613

199.613

62.678

62.678

74,239

73,914

324,987

325,312

Table 5 publishes information about the difference between the average values of the compared groups (Mean

Difference), the empirical value of the Student’s t-criterion (t) and the reached significance level (Sig. 2-tailed).

The trial results show that the mean value of CD4 at the first blood sampling in the main group is 475.26; in the

control group it is 373.32. The difference of 101.935 is not statistically significant, as the achieved significance level

is greater than 0.05 (p = 0.083).

The trial results during the last blood sampling with regard to the average value of CD4 for the main group is 580.65,

while with regard to the control group it is 381.03. The difference of 199.613 proves to be statistically significant,

since the achieved significance level is less than 0.05 (p = 0.002), i.e. the increase of CD4 in the main group compared

to the control group is significant.

• CD8 – First and third date.

Unlike CD4, with respect to CD8 we notice a decrease. Here the null hypothesis (H0) will state that the decrease is

not statistically significant, while the alternative hypothesis (H1) will state that the difference in the mean values

which is observed, is statistically significant.

Table 6 Group Statistics

group N Mean Std. Deviation Std. Error Mean

CD8_1 Case 31 1258,23 707,630 127,094

Control 31 1043,58 610,856 109,713

CD8_3 Case 31 1219,23 603,511 108,394

Control 31 862,35 533,864 95,885

Table 6 contains information on the distribution of the values in the compared groups - volumes (N), average (Mean)

values, standard deviation (Std. Deviation) and standard errors (Std. Error Mean).

Table 7 Independent Samples Test

Levene’s Test for

Equality of

Variances

t-test for Equality of Means

F Sig. t df Sig. (2-

tailed)

Mean

Difference

Std.error

Difference

95% Confidence

Interval of the

Difference

Lower Upper

CD8_1 Equal variances

assumed

Equal

variances

not

assumed

,750 ,390

1.278

1.278

60

58,748

,206

,206

214.645

214.645

167.898

167.898

-121,201

-121,349

550,492

550,639

CD8_3 Equal variances

assumed

Equal

variances

not

assumed

1,609 ,210

2.466

2.466

60

59,120

,017

,017

356.871

356.871

144.717

144.717

67,393

67,305

646,349

646,437

Table 7 shows the difference between the average values of the compared groups (Mean Difference), the empirical

value of the Student’s t- criterion (t) and the reached significance level (Sig. 2-tailed).

Here the result of the average value of CD8 during the first blood sampling in the main group is 1258.23, while in the

control group it is 1043.58. The difference of 214.645 is not statistically significant, since p = 0.206, that is, it is

greater than 0.05. The second half of the Table 7 shows that the difference of 356.871 in the average values between

the main group and the control group in the third blood sampling is statistically significant, that is, the decrease is

significant (p = 0.017).

• CD4/CD8 Case – first and third date

Here, we will apply verification of the hypothesis with dependent samples as it is necessary to follow the change of

the state of the tested indicator under the influence of the particular experimental treatment. The empirical value of

change of the indicators is called an increase in the results. The statistical significance of the increase in the results is

tested by an appropriate statistical criterion for dependent samples.

The null hypothesis (H0) states that the increase in the results is unreliable. To check the hypothesis we use the

Student’s t-criterion for dependent samples.

Table 8 Paired Samples Statistics

Mean N Std. Deviation Std. Error Mean

Pair 1 CD4/CD8_1 ,4642 31 ,25030 ,04496

CD4/CD8_3 ,5594 31 ,27719 ,04978

Table 8 makes characteristic of the distribution of the values of CD4/CD8 ratio during the two dates (the first blood

sampling, and the last), respectively: mean value (Mean), the number of participants (N), standard deviation (Std.

Deviation ) and standard error (Std. error Mean). Table 9 Paired Samples Test Paired Differences t df Sig. (2-tailed)

Mean Std.

Deviation Std. Error

Mean

95% Confidence Interval of

the Difference

Lower Upper

Pair 1 CD4/CD8_1 -

CD4/CD8_3

-,09516 ,07357 ,01321 -,12215 -,06818 -7,202 30 ,000

Table 9 verifies the hypothesis for equality of the average values. The increase in the results is published in the

column Mean, the empirical value of the Student’s t-criterion (temp) – in column t, and its corresponding significance

level - in column Sig. (2-tailed).

The increase in CD4/CD8 ratio for the main group of 0.09516 is statistically significant (p = 0.000).

III. Summarized Results

To establish the effectiveness of the conducted trial we should describe the value and the statistical significance of

the changes that occur in the examined indicators in the presence and in the absence of the experimental treatment.

Figure 4 presents the results of the average values of CD4, which were discussed in the previous section for the

groups – the main group (presence of experimental treatment) and the control group (identical to the main group in

every aspect, except that it does not include the experimental treatment).

Figure 4

•CD4- Control

•CD4-Case

From the figure it is clear that in the beginning of the trial, both groups had similar results; in the course of the trial

both groups increased their values, but the increase in the main group is more pronounced. During the last blood

sampling the main group has significantly higher results in comparison to the control group. All this suggests that the

method used in the main group is more effective.

This is a scientific hypothesis for the verification of which we must pass through the following comparisons:

Firstly, we need to establish the value and the statistical significance of the increase in the results in each group. The

value of the increase is shown in the table as an absolute (d) and a relative increase (d%). The statistical significance

of the increase in the results is established with a parametric criterion for verification of the hypotheses for

dependent samples, made and shown in the previous section. Next, it is necessary to compare the state of the tested

signs in the two groups during each of the tests.

One way to determine the effectiveness of the applied treatment, is to compare the state of the tested indicators in

the end (last date of blood sampling) of the trial. The value of the differences is described by an arithmetic difference

between the average values of the groups, and the statistical significance - again with a parametric criterion for the

comparison of the two independent samples. Summarized information for the development of the indicators under

the influence of the experimental treatment is obtained by comparing the increase in the results for both groups. For

that purpose, we calculate a derivative variable of the difference between the last blood sampling and the first test.

We check the statistical significance of the differences between the increase in the results in the two groups (control

and main) with a parametric criterion for comparison of the two independent samples.

Table 10 presents the results of the processed data for the variable CD4.

Table 10

CD4 N Beginning – first date End – last date Increase Statistical

significance mean mean d d%

Main group 31 475,26 580,65 105,39 22,17

р=0.000

Control group 31 373,32 381,03 7,71 2,07 р=0,704

Difference 101,94 199,61 97,68

Statistical significance р=0,083 р=0,002 р=0,002

The difference between the average values during the first and the last date of blood sampling was presented in the

previous section, as the main conclusion is that an increase of 22.17% in the main group is statistically significant - p

= 0.000. In the control group, the observed increase of 2.07% is not statistically significant (p = 0.704). In this way we

receive the answer of the scientific hypothesis that the treatment applied in the main group is with higher efficiency

than the one applied in the control group.

The comparison of the average values in the groups at the beginning of the study showed that the difference of

101.94 was not significant (statistically significant) - p = 0.083. In contrast, at the end of the trial the difference of

199.61 appears to be significant (p = 0.002). The summarized information for verification of the scientific hypothesis

is obtained by comparing the differences in the increase. In the course of the trial, the main group achieves a

significantly higher increase compared to the control group. The difference between the increase of 94.68 proves to

be statistically significant (p = 0.002).

In an analogous manner, we will address the results of CD8, which are graphically represented in Figure 5 and in

Table 11.

Figure 5

Figure 5 shows the average values at the start and end date of the trial for both groups with regard to CD8. We note

the close values of the two groups at the first blood sampling and the change as a result of the test, which in this

case is the decrease of the average value. We present a scientific hypothesis and apply the same steps, methods and

criteria to establish the statistical significance, just like it was done with respect to CD4 (CD8 is also a quantitative

variable with normal distribution).

Table 10

CD8 N Beginning – first

date

End – last date Increase Statistical

significance mean mean d d%

Main group 3

1

1258,23 1219,23 -39,00 -ЗД0 р=0.529

Control group 3

1

1043,58 862,35 181,23 -17,37 р=0,008

Difference 214,65 356,88 142,23

Statistical significance р=0,206 р=0,017 р=0,114

Table 11 shows the results of the processed data for the variable CD8. The decrease of 3.10%, which is observed in

the main group between the two dates is not significant (p = 0.529). In the control group, however, the decrease of

17.37% is statistically significant (p = 0.008).

As it became clear from the section above, there is a difference of 101.94 in the average values of the groups at the

beginning of the trial, which proves not to be significant (p = 0.203). In the tests at the end of the trial (the last date),

the difference of 356.88 is statistically significant (p = 0.017). Summarized information for verification of the scientific

hypothesis is obtained by comparing the difference in the increase. In the main group we observe a slight reduction

in the increase, unlike the control group, where the decrease is much more. The difference between the increases of

142.23 is not statistically significant (p = 0.114).

Приложение 1

record Date 1 CD4_1 CD8_

1 CD4/

CD8_

1

Date_2 CD4

_2

CD8_

2 CD4

/CD

8_2

Date_3 CD4_

3

CD8_3 CD4/

CD8

_

3

group

212 8.10.15 298 1062 0,28 10.12.15 302 815 0,37 11.2.1

6

367 897 0,41 1

525 8.1.16 340 507 0,67 8.3.16 481 621 0,77 10.5.1

6

452 596 0,76 1

900 7.10.15 276 2017 0,14 14.12.15 337 1972

0Д7 17.2.1

6

311 1716 0,18 1

1409 5.11.15 629 1155 0,54 4.1.16 742 1374

0,54 7.3.15 1112

1718 0,65 1

1473 7.12.15 418 674 0,62 5.2.16 406 599 0,68 14.4.1

6

556 838 0,66 1

1550 22.12.1

5

773 1522 0,51 22.2.16 880 1645

0,54 25.4.1

6

989 1796 0,55 1

1640 7.1.16 287 687 0,42 9.3.16 383 812 0,47 12.5.1

6

350 677 0,52 1

1760 29.12.1

5

315 496 0,64 29.2.16 281 350 0,78 3.2.16 318 425 0,75 1

1790 16.11.1

5

240 530 0,45 25.1.16 421 954 0,44 8.4.16 335 627 0,53 1

1814 2.11.15 845 1898 0,44 4.1.16 860 2158

0,4 7.3.16 952 1978 0,48 1

1834 7.10.15 339 935 0,36 9.12.15 450 973 0,46 17.2.1

6

396 636 0,62 1

1837 12.10.1

5

421 1303 0,32 15.12.15 538 1300

0,41 17.2.1

6

602 1601 0,38 1

1851 9.11.15 580 811 0,72 11.1.16 697 828 0,84 15.3.1

6

577 656 0,88 1

1853 16.10.1

5

597 1170 0,51 22.12.15 595 1055

0,56 22.2.1

6

568 824 0,69 1

1869 16.10.1

5

471 683 0,69 22.12.15 607 852 0,71 29.2.1

6

582 776 0,75 1

1870 16.10.1

5

514 1021 0,5 22.12.15 250 517 0,48 23.2.1

6

455 820 0,56 1

1874 17.12.1

5

712 1173 0,61 16.2.16 450 973 0,46 3.5.16 1043

1468 0,71 1

1878 6.11.15 505 1312 0,39 12.1.16 756 199

7

0,38 14.3.1

6

506 1184 0,43 1

1900 22.12.1

5

997 2692 0,37 25.2.16 854 122

5

0,7 27.4.1

6

1073 1647 0,65 1

1959 4.11.15 295 820 0,36 11.1.16 413 104

8

0,39 21.3.1

6

361 930 0,39 1

1983 26.10.1

5

650 681 0,96 5.1.16 835 826 1,01 7.3.16 836 792 1,06 1

2003 3.11.15 411 878 0,47 5.1.16 445 959 0,46 8.3.16 538 1173 0,46 1

2015 17.11.1

5

567 2255 0,25 19.1.16 571 155

0

0,37 24.3.1

6

502 1727 0,29 1

2049 7.1.16 1261 2818 0,45 7.3.16 966 171

6

0,56 9.5.16 1232 2141 0,58 1

2050 3.11.15 123 2138 0,06 5.1.16 166 180

3

0,09 8.2.16 184 1561 0,12 1

2063 23.11.1

5

116 1539 0,08 20.1.16 122 152

5

0,08 25.3.1

6

160 1876 0,09 1

2072 20.12.1

5

354 2804 ОДЗ 19.1.16 395 270

8

0,15 7.4.16 515 2969 0,18 1

2082 5.11.15 181 1478 0,12 4.1.16 251 175

9

0,14 7.3.16 232 1609 0,14 1

2083 15.10.1

5

258 209 1,23 15.12.15 573 566 1,01 17.2.1

6

595 445 1,34 1

2088 5.11.15 420 778 0,54 11.1.16 413 104

8

0,39 7.3.16 507 717 0,71 1

2122 18.11.1

5

540 959 0,56 19.1.16 510 958 0,53 24.3.1

6

794 976 0,82 1

4 21.8.15 266 659 0,4 20.10.15 256 637 0,4 19.2.1

6

269 269 1 2

43 11.1.16 248 464 0,54 14.3.16 249 389 0,59 16.5.1

5

177 340 0,52 2

71 17.4.15 716 2665 0,27 16.10.15 475 232

5

0,2 20.4.1

6

650 2366 0,27 2

131 12.2.14 140 591 0,24 16.7.15 34 547 0,06 20.5.1

6

132 1281 од 2

150 7.8.15 294 135 2,2 7.12.15 393 224 1,75 5.4.16 370 165 2,24 2

157 19.10.1

5

189 1163 0,16 8.4.16 249 113

3

0,22 8.4.16 257 1299 0,2 2

204 18.9.15 245 790 0,31 16.1.16 328 115

3

0,28 25.5.1

6

268 711 0,38 2

231 14.10.1

5

279 330 0,85 15.1.16 481 478 1,01 12.5.1

6

593 578 1,03 2

339 4.6.15 591 609 0,97 19.10.15 871 920 0,95 7.4.16 684 636 0,92 2

503 15.4.15 731 419 1,75 12.10.15 499 219 2,28 18.2.1

6

502 259 1,94 2

510 18.6.15 288 631 0,46 19.10.15 330 518 0,64 19.2.1

6

360 465 0,77 2

535 5.8.15 144 1400 0,1 7.12.15 149 154

0

од 8.4.16 154 1187 0,13 2

559 2.4.15 270 775 0,35 2.11.15 324 887 0,37 1.4.16 222 508 0,44 2

574 14.4.15 773 1768 0,44 14.10.15 996 204

1

0,49 13.4.1 842 1314 0,64 2

6

621 27.4.15 313 786 0,4 28.12.15 344 889 0,39 19.2.1

6

328 683 0,48 2

679 17.4.15 216 599 0,36 15.10.15 258 822 0,33 15.4.1

6

151 456 0,33 2

884 26.5.15 478 938 0,51 14.10.15 599 1003 0,6 9.2.16 658 1109 0,59 2

917 10.7.15 481 841 0,57 16.11.15 581 1310 0,67 14.3.1

6

499 750 0,79 2

938 10.7.15 225 1153 0,19 8.12.15 226 1111 0,2 8.4.16 110 467 0,24 2

1293 15.10.1

5

231 1010 0,23 16.12.15 272 1340 0,21 19.2.1

6

272 1340 0,2 2

1346 26.5.14 612 889 0,69 14.10.15 723 1129 0,64 4.4.16 541 651 0,83 2

1479 19.6.15 482 1907 0,25 15.10.15 676 2093 0,26 22.2.1

6

578 2256 0,26 2

1483 12.8.15 109 500 0,27 26.1.16 194 629 0,31 21.4.1

6

128 302 0,42 2

1527 4.8.15 761 1607 0,47 4.11.15 632 1246 0,51 1.4.16 544 952 0,57 2

1777 16.6.15 295 1022 0,29 19.10.15 322 845 0,38 10.2.1

6

284 730 0,39 2

1791 14.8.15 408 929 0,44 14.10.15 408 929 0,44 8.4.16 335 627 0,53 2

1854 16.6.15 372 641 0,58 16.10.15 450 609 0,74 16.2.1

6

592 740 0,8 2

2007 14.10.1

5

251 2061 0,12 18.12.15 229 1421 0,16 28.4.1

6

302 1157 0,26 2

2020 26.10.1

5

327 1083 0,3 22.12.15 352 688 0,51 19.2.1

6

346 697 0,63 2

2022 24.8.15 620 1630 0,38 22.10.15 531 909 0,58 25.2.1

6

514 845 0,61 2

2184 4.1.16 218 2356 0,09 2.3.16 157 1819 0,09 9.5.16 150 1593 0,09 2

FINAL REPORT OF THE RESULTS from the clinical, laboratory, hematology and biochemistry tests and from the

monitoring of the vital signs during the respective visits.

Following the dynamics of the relevant parameters showed no deviations from their reference limits, both, in the

main and the control group in all three phases of the clinical trial (See appendixes "2" - hematology and "3" -

biochemical parameters).

During the trial no adverse changes were identified in the somatic status of the patients, neither any clinically

significant abnormalities in the vital signs or adverse side events except for one - a positive dermal test in one of the

patients, due to which he was not included in the trial.

SUMMARY

Results from the immunological tests

• Results from the second test

We observe an increase in the absolute number and the relative share of: the B cells (Wilcoxon's p

the control group it is 1043.58. The difference of 214.645 is not statistically significant, since p = 0.206, that is, it is

greater than 0.05.

The second half of Table 7 shows that the difference of 356.871 in the mean values between the main group and the

control group during the third blood sampling is statistically significant, that is, the decrease is significant (p = 0.017).

Statistically significant decrease of CD8 is as well noted in the control group. The difference in the decrease of the

mean values between the two groups, however, is not statistically significant.

Dynamics in the average values of CD4/CD8 ratio.

The increase in the CD4/CD8 ratio for the main group of 0.09516 is statistically significant (p = 0.000). See Table 9.

Summarizing the results from the present clinical trial, we can draw the following conclusions:

1. The application of the product ITV-1-lmmunH shows good efficacy in patients with HIV-1 infection in its

various stages - in 21 of them (68%) from the main group we note an increase in CD4 + T- lymphocytes; in 16 of them

the increase is accompanied by an increase in the index CD4/CD8 and CD4%, which corresponds to a reduction in the

immune activation. The increase in these parameters is statistically significant compared to the control group. The

absolute number and the relative share of C08 + T lymphocytes is decreasing, too.

2. We observe a very good virological effect – the viral load in 80.5% of the patients is below the threshold of

detection (however, we have no comparison to the control group).

3. The application of the product ITV-1-lmmunH is well tolerated - only one patient manifested allergic dermal

response. With regard to none of them we found variations in the hematological and biochemical parameters,

neither did we establish clinically significant abnormal vital signs.

The Report was prepared by Assoc. Prof. Dr. Ivaylo Elenkov, MD, Chief researcher.

Dated: 23.11.2016

Sources:

Final report of the National Reference Laboratory for Immunology at NCIPD, Assoc. Prof. Dr. M. Nikolova,

PhMD;

Final report of the National Reference Laboratory for HIV at NCIPD, Assoc. Prof. Dr. I. Eleksiev;

Statistical data processing : "Immunotech Laboratories BG" Ltd.

__________________________________________________________________________________

I, the undersigned Mrs. Mariya Krasimirova Foteva, PIN: 8301228618, translator, certify the authenticity of the

translation done by me, from Bulgarian to English, of the attached document: Report, which is composed of 34 (thirty

four) pages.

Translator: ________________

(Mariya Foteva)

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