Penelitian (Research)

Influenza

 virus influenza

We develop vaccine for H5N1 and H1N1 pdm virus, and we develop diagnostic system based on multiplex PCR for H5N1 virus. In developing Influenza vaccine we have grant from Kemenristek Dikti, DIPA Litbangkes, RISBIN Litbangkes. H5N1 vaccines were developed based on isolate that can induce cross protecting neutralize antibodies.  H1N1 pdm vaccines were developed by using consensus DNA sequence based on H1N1 pdm sequence access from NCBI year 2011.

Vaccines were designed based on Hemaglutinin, Neuraminidase and Matriks 1 protein. Hemaglutinin and neuraminidase are two major component of Influenza protein that can induce neutralization antibodies. Matriks is a structural protein that can induce cellular response. There are three kinds of vaccine platform that we develop i.e. DNA vaccine, sub unit vaccine and viral like particles (VLP) vaccine. Each platform can induce immune response in different ways.  Besides that, modifications on vaccine also contribute on the capability of  antigen to induce immune response.

DNA vaccine  can induce both cellular and humoral immunity.  Once deliver into antigen presenting cells, DNA vaccine will reach its target nucleus and followed by antigen expression. The antigen is an endogenous antigen and it will be presented to MHC class I, on other hand the APC expressing antigen can be recognized by phagocytosis cells  and the antigen will be recognize as extracellular antigen that be presented to MHC class II.  Some modification have been done to increase the capability of DNA vaccine to induce humoral  and cellular immune and to change the natural pathway of protein to induce immune response.

Immune response to vaccine based on sub unit protein dominated by humoral response. By adding certain CPP can make sub unit that naturally be processed as exogenous antigen become endogenous antigen and induce cytotoxic response. By using this modification we still can induce immune response like in nature for sub unit vaccine based on structural proteins.

VLP is a structure like virus but do not contain genome. VLP can induce immune response resemble viral infection.  Influenza protein, hemaglutinin, neuraminidase and matriks 1 assemble to form VLP. VLP was develop by using Baculovirus and yeast expression system.

In order to detect cellular immune response, in this case the activity of CD8 cell of vaccinated mouse, we have developed an assay. By using internal ribosomal entry site (IRES) we develop a mammalian expression system that contain Hemaglutinin and eGFP gene which function as marker. The activated CD8 from vaccinated animal destroy cells which expressing hemaglutinin and green fluorescent proteins (eGFP). The activity of CD 8 cells were measured by reduction number of cell expressing eGFP.

Diagnostic system for Influenza especially H5N1 developed to detect the presence of H5N1 specific nucleotide by using PCR multiplex. Primers used in this PCR designed based on specific region of Hemaglutinin, Neuraminidase and Matiks 1. Further, Rapid influenza diagnostic tests (RIDTs) for Influenza A and B, a screening test for influenza virus infection, can be developed. Antigen used in RIDT will be produced in prokaryote. Furthermore, we also has capability to culture influenza virus using cell culture and embryonic eggs as culture media.

Human Immunodeficiency Virus (HIV) virus HIV

            We develop vaccine for HIV based on dominated circulating virus in Indonesia. HIV vaccine platforms are DNA, sub unit protein and VLP. DNA vaccine designed  to induce neutralization antibody to fusion peptide of gp41. We make some modifications to make MPER region of gp41 becomes so more expose that immune cells will recognize it easily.  We also develop sub unit vaccine based on HIV structural protein that fused with CPP that help exogenous HIV protein to become endogenous antigen and will be induce cellular response like in natural infection.   Sub unit vaccine also designed to envelope that expected can induce production of antibody that can neutralize incoming virus or facilitate other cellular response to combat HIV. We also develop VLP that can induce immune response like in viral by using envelope, gag and cellular protein that can enhance B cells response. SF9 cell will be used to produce VLP.

We also develop virus chimera named Simian Human Immunodeficiency Virus (SHIV) that can infect and cause AIDS in Macaca nemestrina.  Some mutations in certain region of HIV gene were conducted in order to gain SHIV. The capability of HIV vaccine either to cure or to  prevent HIV infection will be study using M.nemestrina.

In order to increase the sensitivity and specificity of diagnostic system we develop diagnostic system   based HIV-1 currently circulating in Indonesia.   Diagnostic system that has been established and has been used to monitor HIV-1 drug resistance is HIVDR genotyping. In HIV genotyping we detect mutation at nucleotides coding protease and reverse transcriptase.  Furthermore, we also develop HIVDR genotyping for anti integrase and anti fusion yet still be optimized.

We also produce HIV protein recombinant in prokaryote system. We identified immunodominant peptide of 3 major target HIV proteins i.e. Gag, polymerase, and envelope. The immunodominant peptides of target protein then translated back into codon  by using DNA 2.1. By using same software, we can choose the codons that abundantly presents in E.coli. we use recombinant protein for developing diagnostic system for HIV.  We have some platform diagnostic system for HIV under development, such as avidity assay and Recombinant Immuno blot assay (RIBA).    Avidity assay  is a test used to measure the strength of antigen-antibody interaction. In   new case of HIV infection, the antigen-antibody interaction is weak and be dissociated by using chaotropic agents. Avidity test can be used to measure the success of HIV control program  performed by government.

Basically,  RIBA is similar to HIV western blot. In RIBA, HIV proteins were produced in E.coli, while in western blot, HIV proteins were isolated from virus. RIBA can be used as confirmation test to measure the ‘HIV status’ of patient. Besides that, we develop other diagnostic platform that can be use in HIV screening. Using 3 different combination of HIV-1 protein, we develop either Elisa or lateral flow devices as platform assay for HIV screening.

We develop assay based on nucleic acid test (NAT). One of NATs we develop is kuantitatif PCR (qPCR).  qPCR can be used in detection of viral load in patient. In this assay we design specific primer that could amplify conserved region of HIV, and the usage of specific probe  in qPCR system increases the accuracy of amplification. In order to collect information about HIV genotype that circulating in Indonesia currently, we have started to sequence some HIV gene especially targeted for vaccine and diagnostic development.  Sequencing will be performed by using NGS GS Junior Roche, and now days we have collected sample from at least 5 provinces.

Due to lacking of prove reading capability, reverse transcriptase, an enzyme that change RNA genome of HIV into DNA, could introduce mutation in HIV genome in virus replication. Sometimes mutations on gene coding proteins that are target of action of ARV causes HIV-1 become resistance to ARV.  Furthermore, in the presence of ARV, viruses with no mutation will eliminate, while viruses with mutation will survive and multiply. We try to find out new natural product that has capability to prevent and or inhibit HIV replication, but in this study, we need to strengthen our capability to develop assay that can be used to screening the activity of ARV of natural product.

Human Papiloma Virus (HPV)

virus HPV

HPV is the main cause of cervical cancer,  one of the major health  problems in Indonesia and many countries around the world.  Cervical cancer is one of the fourth deadly female cancer in world. .Even though HPV infection is a self-limited disease that usually clears up without any intervention within several months after acquisition and about 90% will clear up within 2 years,   a small portion of infection   will persist and develop to cancer.  Based on its capability to induce cancer, HPV divide into 2 categories, high risk for HPV causing cancer and low risk for HPV that could not induce cancer.

We develop vaccine based on currently circulating high risk HPV in Indonesia. Designing vaccine that effectively prevent and/or cure cervical cancer must based on nucleotide and amino acid information of Indonesian virus indeed. In this research, we performed genotyping first to find out the type of circulating HPV in women with cervical cancer. Genotyping will be done based  on L1 genes. Then we will perform genotyping for full length HPV categorized as high risk group. Based on genotype information and HLA information (further describe below) we will design vaccine that expected can cure and prevent cervical cancer and HPV infection respectively.  To observed the capability of vaccine to induce immune response we also develop assay for measuring activity of CD8, CD4 and antibody neutralization in animal post vaccination.

 

Human Leukocyte Antigen (HLA) Genotyping

 

Human leukocyte antigen (HLA) genes are located in on the short arm of chromosome 6 (6p21.3). HLA has a critical role in immune responses. HLA is protein that presents self and non self peptide to T-cells, a fundamental step in the initiation of certain adaptive immune response.  HLA only recognizes peptide with certain length and motif.   HLA genes code major histocompatibility complex (MHC) class I and II which activate naïve CD8 and CD4 respectively via MHC-TCR complexes.  HLA is the most polymorphic genes in human. This polymorphism predominantly found within the six classical HLA genes: the class I genes HLA-A,-B and-C and the class II genes HLA-DRB1,-DQB1 and -DPB1.

Polymorphisms is a mechanism to ensure our body can recognize on-going pathogen and eradicate by increasing number of repetitive peptide motifs that bind and present to T cells.  Furthermore, there is an advantage if  people has heterozygote rather than homozygote because heterozygote increases the number of peptide motifs than can be presented to T cell.

To design an vaccine that effectively   prevent and cure diseases caused by infection, we need some information related to  Human Leukocyte antigen (HLA). We will gather information of HLA in normal (that do not infect by certain virus) in person with certain infection such as HIV or HPV.  HLA information can be used for identified epitopes that activate humoral and selular immunity. HLA type determines the faith of epitope to be presented to CD4 or CD8 cells via specific binding with T cell receptor. Effectiveness of vaccine determined not only by epitops that can induce immune respon but also by HLA that will recognize epitop via specific binding  and will present such epitop to CD4 and CD8.

 Vaccine Delivery Systems

 

gene gun

The key of success of DNA vaccine is depended on the capability of DNA to reach its target site, nucleus, where the expression of antigen started. In order to reach nucleus, DNA must overcome many natural barriers such as nuclease serum, cell membrane, endosome, and nuclear membrane. Cell membrane is the outer layer of cell that protect intracellular from unbeneficial environment  Endocytosis is one way by which extracellular substances could pass the lipid bilayer to enter the intracellular environment. Furthermore after endocyted, an extracellular substance will be found inside endosome compartment and designated to be enzymatic  destroyed in lisosom.  The morphology and physiology of nuclear membrane depends on cell development. In dividing cell,  membrane cell is disintegrated and can be found as fragmens at endoplasmic reticulum surround nucleus  and  DNA will enter nucleus passively. On the other hands in non dividing cells, nucleus membran is intact. The transportation in and out of nucleus depends on the capability of extra-nuclear substance to be recognized by nuclear pore through the binding of nuclear localization signal (NLS) found at  extra nuclear substance to specialized cellular protein responsible to nuclear transportation such as impotin.

Natural barier described above will hamper the extra nuclear DNA to enter its target organ. The natural barrier can be avoided by using DNA delivery sistem    There are a lot of DNA delivery system that have been developed. We develop at least 3 kinds of delivery system i.e. peptides, gene gun, and electrophoratore.

Peptide that has capability to deliver other molecules bigger than peptide itself was categorized as cell penetrating peptides (CPP).. CPP also known as protein translocation domain, membrane translocation sequence, or  Trojan peptida. We developed a new peptide-mediated DNA delivery system based on  in silico study. The peptide consists of 3 different peptides that have different function. We identified virus- origin peptide   that has capability to make interaction with cell membrane, bind DNA and function as NLS. The fusion of 3 kinds of peptide will produce a new peptide that has capability to bind DNA, protect DNA from nuclease and deliver DNA into living cells.

We have three peptides candidates that  have capability to binds DNA and protect DNA from nuclease degradation. Based on in vitro study, only one candidate has the capability to deliver DNA pcDNA3.1 eGFP into CHOK1 nucleus that marked by the expression of transgen. Compared to the Lipofectamine, a commercial delivery system, the capability of ALMR to deliver DNA into dividing cell is less efficient, but the capability of ALMR to deliver DNA into non-dividing cells is more efficient compare to Lipofectamine. The addition of ALMR into lipofectamine-DNA complex  could increasing both the percentage of transgene-expressing cells and  viability of cell.

Gen gun was used in plant genetic engineering, now days Gen gun has been used for delivering DNA for human and animal.  Although Gen Gun is effective for delivering gene into nucleus, the apparatus is very expensive.  The DNA must be coated at surface of gold particles which function as bullet by using certain technique.    DNA coated particles will be layers inside the tube; and the DNA coated particles will be push by high pressure to enter the nucleus.   Furthermore once operated, gen gun will produce noise 80-110 decibles that cause permanent hearing damage.    In order to produce home brew Gen gun which is inexpensive, University of Indonesia has been developed portable gene gun which is equipped with silencers, nozzle  and coating apparatus.  In this study we characterize the performance of nozzle   which measured by the capacity of nozzle to deliver material such as ink to penetrate field firing.

We also develop  electroporator.  Once applied on target organ, electroporator induces   short electric impulses that trigger the production of  small pores in the membran. DNA will get into the cell via those pores.  In vivo use of electroporation is done by injecting naked DNA followed by electric pulses from electrode that are located in situ in the target tissues. We develop collaborating with other faculty in Universitas Indonesia, we develop electroporator.

 

Stem cells

 

Firstly introduced by Takashi and Yamanaka in 2000, induced pluripotent stem cells (iPSCs),a method to reprogram somatic cells to become stem cells,  is thought as one of the important breakthrough in the field of stem cell research   along with the earlier isolation of mouse and human ESCs. In stem cells research we concern to develop media that can induce pluripotent stem cells. We develop matrix that function as scaffold that suport the growth of stem cells colonies. We are looking for nutrition from natural product that can induce reprogramming cells. We also express recombinant protein derived from human gene that naturally induce pluripotentcy in embryonic stem cells and can induce reprogramming adult cells to become stem cells.

 

JakCCANDO project (Jakarta CMV, Cardiovascular, Antiretroviral, Neuropathy, Dental, Ophthalmology)

JakCCANDO

This is a collaboration project between Universitas Indonesia and University of Curtin, Australia, under direction Prof Patricia Price. This project  investigate the recovery of immune function and the role of cytomegalovirus (CMV) in patients recruited and monitored in Indonesia. Preliminary data shows antibody responses to HIV are extremely high in Indonesian patients with a significant (but undocumented) burden of disease. A team of physicians and scientists is assembled for this work, with sample archives, detailed databases, laboratories and techniques arranged.
Evidence of repeated CMV reactivation (the “footprint” of CMV) will include CMV DNA (by PCR), antibodies and CD8 T-cell responses. This will be correlated with:
a) immune activation, immunosenescence and T-cell homeostasis
b) clinical outcomes previously associated with CMV in other contexts, specifically ocular, cardiovascular and neurological sequelae assessed by expert physicians
Natural killer cells will be considered as a mechanism affecting the “footprint” of CMV, including an evaluation of the importance of genetically-determined variations in NK activity. Methods for assessment of these functions are established.