Material and Methods:
1.6.1. Cells, plasmids and antibodies
116 cells will be used in construction and amplification of helper-dependent adenoviral (HDAd) vectors. 116 cells will be kept in complete Dulbecco’s modified Eagle’s medium (DMEM) and boosted with 1% penicillin-streptomycin to 100U/ml and L-glutamine to 2 mM, 0.01mg/mL of hygromycin and 10% heat-inactivated fetal bovin serum (FBS) in a 37oC 5% CO2 incubator. HDAd plasmids that are encoded to broadly neutralizing antibodies will be used as well as helper virus and PmeI restriction enzymes.
1.6.2. Construction Helper-Dependent Adenoviral (HDAd) vectors
In general, HDAd vectors will be produced by using the Cre/loxP system. The helper-dependent adenoviral vector will be produced as a bacterial plasmid. By using standard cloning techniques, the transgene-encoding expression cassette will be made and ligated into the pStuttle vector. HDAd genome will be linearized by I-Cell and PI-Scel restriction enzyme for endonuclease sites that are found immediately next to the ITRs. According to the manufacturer's instruction, plasmid purification kits will be used to purify HDAd plasmid. Polymerase chain reaction (PCR) will be performed with specific primer DNA with the principle’s listed thermocycling condition to produce gel-purified. After using the restriction endonuclease to digest HDAd, ligation will be conducted at 16OC through the night and changes will be performed with E. coli.
1.6.3. Amplification and Purification of HDAd38
Statement of project:
HIV is a lentivirus that infects human in several ways such as blood transfusion, pregnancy, and sexual contact. HIV causes HIV an infection that leads to acquired immunodeficiency syndrome (AIDS). There is no current remedy or vaccine for HIV. Patients with HIV are treated by an antiretroviral therapy (HAART) that is highly active. Helper-dependent adenoviral HDAds are modified vectors that are deleted all viral coding sequences, which increase the capability to transduce a wide range of cells. Broadly neutralizing antibodies protects cells from antigens by neutralizing biological effects. Broadly vectored neutralizing antibodies attack gp120 to prevent HIV-1 acquisition in humanized mice. We have determined that HDAd vector could express a high level of bNAbs in NGS-Hu PBL which could increase T helper cells (CD4+) titer and reduce the level of HIV RNA in plasma. In this study, we will test if vectored bNAbs can suppress or significantly delay viral rebound by shrinking the size of the persistent viral reservoir, allowing for prolonged, ART-free remission in NGS-Hu PBL mice.
1.2. Introduce to HIV:
The human immunodeficiency virus (HIV) falls in the lentivirus group, which is a subgroup of the retrovirus. HIV causes HIV infection which leads to acquired immunodeficiency syndrome (AIDS) in the late stages. AIDS is a disease that causes failure in the immune system leading to opportunistic infections and cancers. Without treatment, patients with HIV infection have an average survival rate of 9 to 11 years1. HIV can be transmitted from one person to another through sexual relations, blood transfusion, sharing contaminated needles, breastfeeding and during pregnancy for an infant. HIV can be transmitted through sex by contact with blood, semen and vaginal fluids. An infected mother can infect her infant through breastfeeding. In addition, the infant can be infected from his mother during pregnancy and childbirth because of exposure to mother’s blood or vaginal fluid. HIV is present in these body fluids as free virus particles and virus inside infected immune cells2. HIV originated from non-human primates in West-central Africa and the early 20th century was transferred into humans. HIV-1 was discovered in Cameroon during diagnosis a simian immunodeficiency virus (SIV) in wild chimpanzees (HIV-1 descends from the SIV). People who participate in bush meat or wild meat activates usually acquired SIV. SIV is a weak virus and can be destroyed by the immune system, but through several transmissions of the virus from human to human leading it to mutate into HIV3.
1.2.1. HIV Prevalence:
HIV differs in prevalence from country to country. At the end of 2016, there were 36.7 million people around the world who were infected with HIV.1 million of these were children < 15 years old. Today, only 60% of patients with HIV know that they have HIV infection while 40% still need to access to HIV diagnostic tests. In addition, 20 million patients with HIV have access to antiretroviral therapy. The number of people who have been diagnosed as HIV patients since the start of the epidemic is 78 million. The total number of people who died from AIDS-related diseases since HIV discovered was about 35 million which means there were 1 million people who died every year from the AIDS-related disease. The majority of people who have HIV infection live in low and middle-income countries. Most people with HIV are living in sub-Saharan Africa by 25.6 million which represent about 66% of HIV patients around the world. In 2016, the cases of new HIV infections decreased to 1.8 million cases compared to 2.1 million in 2015. In 2015, new HIV infected children decreased by 50% compared with 2010 because of accessing about 77% of pregnant women to antiretroviral therapy which prevents transmission of HIV infection to their babies. South Africa has the highest number of people with HIV which represent over 10% of all people living with HIV in the region. Prevalence of HIV in Swaziland is the highest by 27.20% then Lesotho by 25% then Botswana by 21.90%. from 36.7 million people with HIV around the world, the highest number of these people are living in South Africa by 7.1 million, followed by 3.2 million in Nigeria and 2.1 million in India4. There are several methods to prevent HIV infection such as using a sterile needle, safe sex, breastfeeding and treating infected people2. HIV has flu-like symptoms which cannot be specific for diagnosis HIV. However, several laboratory tests can diagnose HIV for sure. Although treatment with antiretroviral can slow the course of the infection and it is recommended as soon as it is diagnosed, there is no cure or vaccine can prevent HIV. Patient without treatment may survive 11 years after infection5.
1.2.2. Clinical Diagnosis:
Physicians cannot rely on symptoms of HIV to diagnosis patients because the only way to make sure that a patient has HIV is to get tested. The symptoms of HIV are different and dependent on the patient’s stage of HIV infection. People with HIV infection in early stages (acute stage) have flu-like symptoms in weeks after HIV infection. A few people with HIV have no symptoms during early stage. Symptoms in the early stage of HIV are: fever, night sweats, rash, chills, muscle aches fatigue, sore throat, mouth ulcers, and lymph nodes endearment. After the early stage of HIV infection, patients enter to latency stage also called “chronic HIV infection.” Patients in this stage have no symptoms related to HIV even HIV is still active but reproduces at a very low rate. The last stage is the progression to AIDS when the virus destroys the body’s immune system5. Symptoms in this stage can include rapid weight loss, extreme weakness, prolonged lymph nodes enlargement, pneumonia, diarrhea, blotches on or under mouth skin, memory loss and sores of the mouth, anus or genitals.
1.2.3. Laboratory Diagnosis:
Laboratory tests are the most accurate way to diagnosis HIV, and it is commonly diagnosed by testing blood or saliva. The enzyme-linked immunosorbent assay (ELISA) is the first screening test that was used to diagnose HIV and is still used because it is very sensitive. If an individual has HIV, his serum will depict the presence of antibodies which will get attached to the HIV antigens that are in the plate. Secondary antibody will be added to bind to human antibodies and change in color in present a substrate with considering washing between steps, then ELISA result will report as a number. If the results of an ELISA test come out to be positive, the results are confirmed by the use of the Western blot test. The Western blot is a test that is used to detect antibodies, and it is used to confirm the diagnosis of HIV after a positive result for ELISA. In the western blot, proteins that are produced from the HIV- infected cells will be separated and immobilized on a membrane. Then, the primary antibody is added to bind to HIV antigen during incubation. Washing is applied to remove free antibody then adding a secondary antibody that is linked to an enzyme signal. The secondary antibody will produce a signal that indicates the presence of HIV antigen6. There is a home test approved by FDA in 2012 as a rapid test for diagnosing HIV. This test needs a drop of blood or saliva to give a primary result. If the result is positive, the person needs to see a specialist doctor to confirm the diagnosis. However, if the result is negative, it needs a few months to repeat the test to confirm the result7. Moreover, the antigen test, p24, is a test that reacts to the availability of the p24 protein that is the capsid protein of HIV. P24 antigen test is used to diagnose HIV in early detection because the p24 antigen increases soon after infection. Specific monoclonal antibodies for the p24 antigen are mixed with the patient blood. In present HIV, monoclonal antibodies will bind to the p24 antigen and an antibody linked to an enzyme, to the monoclonal antibodies leading to a change in color (IOTF,2000). The P24 antigen test is no longer used in the United States for screening blood donation because of the risk of false negative results during the window period. Nucleic acid testing (NAT) can be more accurate than p24 antigen test for screening blood donation8. The test called Polymerase chain reaction (PCR) is used to determine the HIV RNA through extracting the RNA from the plasma of the patient, which is later treated with the enzyme reverse transcriptase to change the HIV RNA to DNA. Then, PCR processes are applied to amplify the DNA and finally the amount of virus in the patient’s sample can be counted to detect changes9.
1.2.4. Treatment:
Currently, no cure is known to treat neither HIV nor a vaccine to prevent it. HIV is treated by the antiretroviral therapy (HAART) that is highly active, which reduces the gradual development of HIV in the body of the individual. In addition, treatment prevents opportunistic infections that are caused by immunodeficiency. The highly active antiretroviral therapy (HAART) contains at least three medications belonging to antiretroviral agents. Two nucleoside analog reverse transcriptase inhibitors and a non-nucleoside reverse transcriptase inhibitor are used to form the HAART cocktail. According to WHO, antiretroviral therapy is recommended for patients with HIV at any age as long as the virus is detected in the body of the individual. The therapy is also recommended for pregnant women even without regard for the CD4 count. Treatment requires to be continued without a break when it is started. The main objective of antiretroviral therapy for the long – term is to lower plasma HIV-RNA count below 50 copies/ml. After four weeks of treatment, it is recommended to measure the level of HIV-RNA copies to determine the effectiveness of therapy then checking every three to six months. If the level of HIV-RNA copies is below 50 copies/ml, the treatment is effective. Antiretroviral therapy is effective in more than 95% of patients during the first year. HAART can decrease the risk of development to AIDS and death as well as improve physical and mental health10. Additionally, treatment of HIV decreases 70% of the risk of acquiring tuberculosis11. Moreover, the benefits of treatments include reducing the risk of HIV transmission through sex and in pregnant women to their children. Compliance of treatment is the key to ITS effectiveness. However, the lack of access to medical care in some areas is the reason for non-adherence. Side effects of antiretroviral therapy include dyslipidemia, lipodystrophy syndrome, diarrhea, the risk of cardiovascular and diabetes mellitus11.
1.2.5. HIV Biology:
The HIV genome and proteins are interesting topics in the research field since the discovery of the HIV in 1983. The virus contains a matrix and a viral envelope that encloses a capsid that includes two copies of the single strand RNA and many enzymes. HIV itself was discovered after two years from registering the first case of the disease. HIV has a different structure from other retroviruses and its diameter about 100 nm. The inner structure for HIV contains a core that is shaped like a cone which contains two copies of the single strand RNA, the reverse transcriptase enzymes, protease, integrase, minor proteins, and the major core protein. There are 16 viral proteins that play important roles in the HIV life cycle. HIV-1 has two copies of positive-sense ssRNA that is enclosed by a conical capsid that contains protein p24 as well as two copies of noncovalent bond12. The strand of RNA in HIV contains 9749 nucleotides as long with a 3’ poly(A) tail, a 5’ cap, and various reading frames. The ssRNA binds to p7 nucleocapsid protein, protein p6 and critical enzymes that are important to the development of virus-like reverse transcriptase and integrase. The nucleocapsid cooperates with the genomic RNA to prevent the RNA from breaking down by nucleases. The RNA is also surrounded by different particles such as viral infectivity factor (Vif), Vpr, negative regulatory factor (Nef) and protease. Viral infectivity factor is a protein responsible for disrupting the activity of antivirus of enzyme APOBEC. Vpr is a human immunodeficiency virus gene, and Nef is a small protein that is encoded by primate lentiviruses. The matrix of protein p17 encloses the capsid to protect the virus components. This matrix is surrounded by a cover of the host cell that is created when the capsid grows from the host cell by acquiring some membrane from it. This envelope contains glycoprotein 120 (gp120) and glycoprotein 41 (gp41) that are used for binding and inserting the virus to the host cell. Medical scientists work on targeting gp120 and gp41 by vaccines to prevent HIV infection because gp120 and gp41 are the only proteins on the top membrane of the virus13.
1.3. Helper-Dependent Adenoviral:
Helper-dependent adenoviral vectors (HDAd) are characterized by the fact that they do not have all viral coding sequences that can increase or capably convert different types of cells from different species that depend on the cell cycle. In addition, HDAd vectors are characterized by long-term transgene expression. HDAd can effectively correct genetic disorders in many animal models without causing chronic toxicity 16. One of the most important advantages of HDAd vectors compared to the other types vectors is that HDAd vectors have a large cloning capacity that up to 37 kb. The large cloning capacity of HDAd allows them to deliver more therapeutic genes and wide cis-acting elements as well as genomic loci. Cis-acting elements work on regulating and prolonging transgene expression. The HDAd genome is dependent on the nature of the stuffer sequence17. There is no evidence that HDAd has relation with the risk of carcinogenesis, especially insertional and germ-line transmission18. In addition, producing high-quality HDAd can be effective for tasting on animal models and maybe human clinical trials19. Deleting all viral gene sequences from adenoviral and leaving cis-acting elements that are necessary for replication of vector genome (ITRs) and encapsulation is the easiest way to avoid the immune system especially adaptive response against Ad vectors. Because of deleting all viral gene sequences in Ad, the vector cannot be amplified by itself. Therefore, Adenoviral vectors depend on a helper virus (HV) for propagation, therefore, they are called helper-dependent adenoviral vectors (HDAd). In this way, helper virus must have normal replication and all viral proteins expression that required to package and replicate the HDAd genome. However, it is important to limit infectious HV propagation to achieve a relatively chaste HDAd. Kochanek is one of the most effective methods to prevent propagation of HV through producing HDAd12.
1.4. Broadly Neutralizing Antibodies:
Neutralizing antibodies are antibodies that protect cells from antigens by neutralizing biological effects. Broadly neutralizing antibodies (bNAbs) target various strains of a specific virus like HIV. The first bNAb (b12) was discovered in 1991 and development of this type of antibodies have been increasing in recent years21. bNAbs are shaped by mutations that are located at the upper ends of the Y-shaped antibody that has loops to contact viral epitopes as well as they are stickier than other antibodies. The flower-like envelope protein on the HIV-1 surface like gp120 and gp41 are the most sites for bNAbs. These glycoproteins sprout from HIV membrane and its function to grab and attach host cells 22. Broadly neutralizing antibody is an effective strategy to prevent HIV by responding against the envelope protein 23. approximately 1% of infected HIV-1 patients produce these broadly neutralizing antibodies against HIV-1 in order to initiate an immune response that is initiated because of a significant number of viral variants, continuous viral replication, and instable antigen exposure24. BNAbs can be synthesized by intermuscular blood injection that provides antibodies at specific concentration levels that may able to prevent HIV-1 acquisition in both blood and mucosa25. Anti-HIV bNAbs can be classified into two types which are first and second generations26. The differences between these two groups are their functions and the method that used to generate bNAbs. For instance, first-generation antibodies such as b12,2F3, 4E10 and 2G12 they were constructed from Epstein-Barr virus (EBV) B cells that continue undergoing mutation for using phage- display methods. Even though these first generation bNAbs provide 50% to 80% of neutralization breadth, they are not the best option for an antibody-based vaccine for HIV-1 because of low efficacy to neutralize and a limitation of the method (Antibody specificity selection and limitation of screening option)27. However, the second generation of bNAbs shows high flexibility in technology manipulation and improvement in neutralization potency. Technology provides a new selection and screening method as well as antibodies analysis and isolation method28. There are several second-generation bNAbs such as PDGM1400, VRC01, PG9, PG15, PGT121, PGT145, CH01 and CH103. To date, there is no any bNAb gets the FDA-approved to use in HIV treatment. The improvement of bNAbs as therapeutic antibodies against HIV is undergoing, and several of these bNAbs have successfully passed to human clinical trials 29.
1.5. Rationale
From the literature, it is clear that HDAd may help to produce anti –HIV broadly neutralizing antibodies (bNAbs). These monoclonal antibodies can recognize HIV membrane component gp41 and gp120 and target them. Broadly neutralizing antibodies (bNAbs) characterized by some uncommon features such as auto and polyreactivity and a high degree of somatic mutation as well as extended CDR H3 regions. These unusual characteristics help bNAbs to increase the probability of avoiding immune system response30,31,32. Therefore, using viral vectors to transfer of bNAbs has been investigated. This type of gene therapy may need less frequent dosing. Previously, adeno-associated viral (AAV) vectors were used to carry antibody-like immunoadhesins to provide long-term protection against simian immunodeficiency virus (AAV) and sustain gene production33. Even though intramuscular injection of AAV- vectorized bNAbs provided protection against intravenous and made mucosal problems with HIV in humanized mice, some limitations included problems in virus propagation and purification as well as a small transferring capacity of 4kb34, 35, 36, 37. In 2015 another study in Dr. Kumar lab that is applied on broadly neutralizing antibodies that are encoded in first-generated adenoviral (ADV) vectors. This study showed that a single intramuscular injection bNAbs that are encoded on FG ADV vectors in humanized mice could produce a high level of broadly neutralizing antibodies (bNAbs) in the serum which led to protect against many repeated problems with a relatively high dose of HIV-1. In addition, this single intramuscular injection of ADV-vectorized bNAbs prevented reduction of peripheral CD4+ T cells as well as declined the level of HIV in plasma below detection limits. However, FG ADV vectors have a limited time of transgene expression by about six weeks. Additionally, compared with HDAd vectors, FG ADV vectors have limited the capacity by 5-8 kb38. However, the probability of prophylaxis and treatment of HIV by using HDAd encoding anti-HIV broadly neutralizing antibodies is unknown. This study aims to test whether vectored bNAbs indefinitely suppress or significantly delay viral rebound by shrinking the size of the persistent viral reservoir, allowing for prolonged, ART-free remission in a humanized mouse model. To answer these questions, before and after treatment humanized mice with vectored bNAbs we will:
A. Constriction, amplification and purification of HDAd vector to be encoded by bNAbs to prepare them for injection into humanized mice.
B. HDAd injections into humanized mice to produce bNAbs against HIV-1
C. Measurement of mouse- generation bNAbs to observe the amount of bNAbs in the mice serum.
D. Viral load assessment to identify the quantity of plasma HIV RNA.
1.6. Material and Methods:
1.6.1. Cells, plasmids and antibodies
116 cells will be used in construction and amplification of helper-dependent adenoviral (HDAd) vectors. 116 cells will be kept in complete Dulbecco’s modified Eagle’s medium (DMEM) and boosted with 1% penicillin-streptomycin to 100U/ml and L-glutamine to 2 mM, 0.01mg/mL of hygromycin and 10% heat-inactivated fetal bovin serum (FBS) in a 37oC 5% CO2 incubator. HDAd plasmids that are encoded to broadly neutralizing antibodies will be used as well as helper virus and PmeI restriction enzymes.
1.6.2. Construction Helper-Dependent Adenoviral (HDAd) vectors
In general, HDAd vectors will be produced by using the Cre/loxP system. The helper-dependent adenoviral vector will be produced as a bacterial plasmid. By using standard cloning techniques, the transgene-encoding expression cassette will be made and ligated into the pStuttle vector. HDAd genome will be linearized by I-Cell and PI-Scel restriction enzyme for endonuclease sites that are found immediately next to the ITRs. According to the manufacturer's instruction, plasmid purification kits will be used to purify HDAd plasmid. Polymerase chain reaction (PCR) will be performed with specific primer DNA with the principle’s listed thermocycling condition to produce gel-purified. After using the restriction endonuclease to digest HDAd, ligation will be conducted at 16OC through the night and changes will be performed with E. coli.
1.6.3. Amplification and Purification of HDAd38
116 cells will be kept in 66-mm dishes in complete DMEM with heat-inactivated FBS to 10%hygromycin and 1% penicillin-streptomycin and L-glutamine to 2 Mm. 116 cells will be split in 6 well plates (plates size increase with increase HDAd titer) every two days (until they reach 90% confluency). The split cells will be incubated at 37oC 5% CO2 to warm media before they are removed from the medium, use PBSto refresh 116 cells and trypsin to detach cells then the cells will be re-suspended with complete DMEM in 6 well plates. The 116 cells will be transfected with HDAd and HV in passage 0 then co-infected every two days with the helper virus (HV). For co-infection, appropriated amount of HDAd and HV (Diluted with PBS++) will be added to cells after removing media then incubate for 30 minutes at 37oC and 5% CO2. Low serum H+ DMEM will be added to infected cells then incubated for two days. The titer of the HDAd is expected to increase by subsequent coinfections of 116 (293Cre) cells with the HDAd and the HV. After several amplifications (Passages) in 6 well plates and 60 mm plates, Large-scale will be used to produce large quantities of HDAd. HDAd amplification is monitored by using the tittering process that includes 293 cells, fixation buffer in 96 well plates, passages dilution from10 to 100-fold until maximum pack will be achieved and Lac Z mixture adding to count blue-forming units (BFU). Finally, HDAd will be purified by using CsClultra centrifuge then adding glycerol to a total concentration of 10% for long-term storage.