A Holistic Introduction to Truvada for Pre-Exposure Prophylaxis And PrEP as a means of combating the HIV epidemicIntroduction: The purpose of this essay is to introduce Truvada for Pre-Exposure Prophylaxis “PrEP”, a treatment measure being used to prevent viral infection of non-infected individuals exposed to HIV. This essay will discuss the process of an HIV infection, the viral replication processes Truvada targets, the stages of a HIV infection’s progression to AIDS and the challenges faced in finding an expansive cure for HIV. Secondary questions answered in this essay include side effects of the medications, the most effective way of taking the medication, current methods for treating HIV and other proposed and tested methods for combating the disease.A. Stages of HIV Infection  HIV is divided and classified into two forms HIV-1 and HIV-2, based on genetic differences commonly related to geographic occurrence. Both forms of the virus can be further divided into subtype: groups or clades originally highly related to the density of geographical occurrence. It is important to establish what form and subtype of the virus is present in an infection in order create a medication cocktail efficient in addressing infection (Aids Map).HIV infection exists in the 3 main stages, the 1st stage is known as the acute infection stage usually lasting anywhere from 3-6 weeks, with patient’s experiencing vague, broad ranging symptoms. The CD4 cell count plummets while viral particles increase and CD8 antibody cell count rises as the immune system mounts a response. The early detection test for acute infection is the p24 antigen test using ELISA, which recognizes the presence of viral proteins in the blood only during the first few weeks of infection. If positive or negative and in the window period of exposure, the presence of viral proteins in serum is double-checked using a Western blot (Khan Academy – Diagnosing HIV). As the HIV antibody levels match the production of viral particles the spike in viral particles and the initial drop in CD4 cells begins to stabilize as the immune response begins to take effect. The production of viral particles is limited and the CD4 count is able to regain higher, but lower than normal, levels.At this point the infection enters the 2nd stage known as chronic infection where the virus slowly depletes the CD4+ cell count in the body overtime if left unchecked. The majority of CD4 cell death occurs from natural the immune response calling for cell destruction. This initializes mechanisms of pyroptosis and apotosis in order to kill off infected cells and surrounding cells with the potential of becoming infected. Only 5-10% of cells that die off are infected, the remaining 95-90% of cell death is in response to immune inflammatory response signaling (Khan Academy – How HIV kills). When the CD4 count is less than 200 copies per cubic mm compared to the normal CD4 cell range of 500 to 1400 copies per cubic mm blood an HIV infection has progressed to Acquired Immunodeficiency Syndrome (AIDS). The weakened immune system allows for opportunistic infections such as pneumonia, flu and other bacterial or viral infections to take place. In order to combat this progression, researchers have developed antiretroviral medications and therapy routines to limit the latent destruction of the immune system overtime.B. HIV Entry and Replication in CD4 cells The human immunodeficiency virus is a retrovirus that attacks the immune system by depleting the number of CD4 cells in the body.  Entry into CD4 cells can be broken into five steps. Frist a viroin must enter the body and attach to a host CD4 cell, this attachment is fairly nonspecific making it easy for HIV to interact with a target cell. HIV uses Env a surface glycosylated trimer of gp120 and a heterotrimer gp4 (NCBI – Arrildt, Joseph, Swanstrom). HIV infects CD4 cells by fusing with cell membrane using the CD4 receptor and a co-receptor either CCR5 or CXCR4. Through conformational changes in the variable loop domains of gp120 a bridging sheet is formed between the viroin and the CD4 membrane. This is followed by the viroin binding with either co-receptor and the movement of the viroin to a location on the membrane it can use for productive fusion. The binding of the co-receptor allows for the gp41 peptide to act on the CD4 membrane and viral membrane brining them in close proximity to each. The energy garnered from this rearrangement of peptide alpha helices provides the energy for fusion pore formation (TRACO 2015 – 1:15). This allows for a fusion pore to form leading to inject of an mRNA template and viral replication enzymes into the cytoplasm of the cell (NCBI – Arrildt, Joseph, Swanstrom).Once inside the cell the virus template can use mRNA to and reverse transcriptase to make a cDNA to the template strand. Up until this point HIV has not infected a living CD4 cell, it has only entered the cell and primed itself to take over cellular machinery to replicate and make more viral copies. ~ This is a key step in understanding the virology of HIV. Particularly in the case of retroviruses; where a virus hijacks the genetic material and machinery of a cell making it incredibly difficult to isolate and destroy in the body. If the infectious process and proliferation of viral particles can be understood in discreet detail, specific techniques to interrupt these steps can be useful in preventing viral infection. ~ When a cDNA strand is made, viral integrase brought in with the prointegration viral complex, can bring the newly synthesized genetic material into the nucleus and with various transcription factors incorporate viral DNA into the cells DNA. This step is another critical point in order for the virus to establish infection. The infected cell can now take two pathways:(1) The virus can hijack cellular machinery to produce more viral proteins and mRNA to make new viroins that will eventually bud off from the initially infected cell and infect surrounding CD4 cells.(2) The virus can lay dormant as a provirus in the host DNA. This is key for establishing chronic infection and is the challenging obstacle for researchers in addressing HIV infection. (2018 Demystifying Medicine)(NIAID – Replication)  C. What is Truvada and how does it work?Truvada is the brand name for two medications initially prescribed for Post-Exposure Prohylaxis now being using in a drug pre-exposure method of preventing HIV from establish infection. In combination Truvada is a blue pill containing 200 mg of Emtricitabine and 300 mg of Tenofovir Disoproxil Fumarate (TDF), which is converted to Tenofovir in vivo. Both drugs are nucleoside reverse transcriptase inhibitors nRTIs. In the event that a viroin is able to deliver viral protein including protease, integrase and reverse transciptase into a CD4 cell these medications act as inhibiting analogs that attach to the reverse transcriptase enzymes of HIV-1 (HIV-1 RT) to prevent it from acting on viral RNA to make cDNA. Emtricitabine is a nucleoside analog of cytidine  and Tenofovir is an acyclic nucleotide analog of adenosine 5’-monophosphate (FDA access data). Both drugs act on the process of reverse transcriptase via chain termination. TDF is converted in vivo to Tenofovir disphosphate by diester hydrolysis and phosphorylations by cellular enzymes.  Tenofovir diphosphate is now able to compete with the natural substrate deoxyadenosine 5’-triphosphate and inhibits the activity of HIV-1 RT after being incorporated into the DNA resulting in chain termination. Emtricitabine is first phosphorylated to Emtricitabine 5’-triphosphate, which acts is similar mechanism, but competes with the naturally occurring substrate deoxycytidine 5’-triphosphate. It is incorporated ino viral DNA and results in chain termination. Both nRTIs have weak binding effects to mammalian DNA polymerase and mitochondrial DNA polymerase, making them suitable drugs for targeting viral DNA replication (FDA access data).In the strong probability that the viral mechanics are halted here the virus has not established infection, by not being incorporated in the host’s DNA, a key step in establishing acute and chronic HIV infection.  In pre-clinical trials, Truvada is seen to be most effective when take daily as an oral tablet, with dosages differing in accordance to age, body weight and medical clearance (Rx-List – Truvada). By doing so blood serum levels are able reach drug levels proven to hold a greater than 90% chance against infection. HIV initially enters the body via transcytosis of mucosal membranes. In rectal tissue it takes 7 days of taking the medication to reach effectiveness. In vaginal tissue is takes 20 days to reach effectiveness.D. Potential side effects and blood tests while taking Truvada As with many synthetic drugs Truvada has a number of short term and rare long term side effect that should be monitored over the course of taking the drug.Some of the short term side effects of Truvade include: Nausea, Headaches, Dizziness, Weight loss, Stomach pain, Vomiting, Hematuria, Urine production, Joint pain, Back pain.Some of the long-term side effects of Truvada include: Mild to moderate kidney impairment and bone marrow density loss. Standard blood tests measuring kidney functioning should be taken every 3 months while on PrEP to monitor kidney function while on PrEP. No specific reasons have been targeted as to why some individuals are able to tolerate the medication with renal impairment and while others are not. In addition, standard sexually transmitted disease testing should be performed with standard blood work for PrEP given the increased rate of sexually transmitted infections with the clinical usage of PrEP.E. Antiretroviral Therapy (ART) and Truvada in relation to other antiretroviral drugsIn 1987 Azidothymidine was the first antiretroviral drug used in clinical treatment of HIV. As a drug it was first used to combat cancer and in lab studies it was seen to have suppression effects against HIV cells, without damaging normal cells. Aziothymidine is also an nRTI. In the beginning of the 1990s other antiretroviral drugs were being developed. Due to the high rate of replication error ~ mutation of HIV, using one drug as a treatment regimen was losing its level of effectiveness. New nRTIs were developed as used in a dual drug therapy model as an improved means of suppressing viral progression. The development of new classes of antiretroviral drugs protease inhibitors led to the development of triple drug therapy known as Highly Active Antiretroviral Therapy.Protease inhibitors act on suppressing infection after the virus has infected a host cell. Should the virus take an active course it translates metabolites and viral peptides using the cell’s machinery. Protease aids in viroin maturation and is responsible for cleaving the viral metabolites and particles developed in packaging. Peptides must be cleaved in order for them to be active in viroins budding off from the dying host cell. By limiting this step the virus has a lessened ability to infect neighboring cells and increase viremia. Some protease inhibitors used in treatment today include: Atazanir, Darunavir, Fosamprenavir and Indinavir.The development of antiretroviral drugs was a tremendous in combating the HIV Pandemic at that time. Today scientists have developed drugs in anticipation of the rapidly mutating virus. Therapy treatments are designed to combat different mechanisms of the virus to suppress it at different points in its replication process. Approved classes of ART drugs include:Entry InhibitorsFusion InhibitorsReverse Transcriptase Inhibitors (nucleoside and non-nucleoside)Integrase InhibitorsProtease Inhibtors(An extensive of FDA approved drugs with brand names can be found at: https://betablog.org/haart-chart/(Aids Info –NIH)F. Treatment methods and challenges around curing HIVAs seen in the diagram above researched have managed to target specific points to interrupt viral progression. With HIV’s rapid replication rate of 24 hours scientists and clinicians have been strategic in pairing drugs that address suppress viral progression prior to steps allowing for viral mutation (TRACO 2015 – 1:16).  This is best witnessed in the viral colony phylogeny trees developed from RNA assays of patients prior to and post ART treatment. Trees show viral suppression limited to one common ancestor as viral RNA assays are taken over time (2018 Demystifying Medicine). This is an important featuring in understanding the persistence of an HIV infection. Chronic infection is not due to the mutation rate or proliferation abilities of the virus overtime. Chronic infection is maintained by HIV DNA reservoirs of latent integrated virus over time. Though the number of viral copies in the blood can reach undetectable levels recognized as less than 20 copies of virus per mL of blood the infection remains present as dormant integrated DNA in blood serum and lymphocytes that was established prior to ART therapy. The immune system does not recognize latent provirus until the production of viral peptides is initiated. This is the difficult step is curing an infection.Researchers at pharmaceutical company Calimmune have attempted to stimulate and eliminate latent viral particles using latency reversal agents and then injecting tissues with antibody targets to Env expressing cells. However, the window of timeframe for this as effective measure in curing the disease has been proven to be too short to maximize immune effectors (2018 Demystifying Medicine). Researchers at pharmaceutical company Sagamo have also tried gene therapy by limiting a viroin’s ablity to fuse with cell during the co-receptor step. As previously mentioned HIV viruses can use one or both of two known CD4 co-receptors. However is there is a dysfunctional protein receptor the virus cannot properly bind to a CD4 cell, preventing viral contents from entering the cell. This dysfunction protein receptor is translated by the mutant CCR5 delta-32 gene, which is rare to fine, but present in some individuals. By knocking out the CCR5 gene ex vivo and re-infusing patients the ability of a more commonly diagnosed HIV-R5 to enter a CD4 cell is limited. If the virus contains the CXCR4 receptor domain then it can fuse with the cell and initiate infection (NCBI – Lopalco). In line with gene therapy and still being studied is the possibility of using CRISPR/Cas9 and the concept of endonucleases to target viral DNA and cleave it from host cells to insert non-viral DNA in its place. (CRISPR/Cas9) G. Social factors limiting the public health efforts regarding PrEP When prep was first released there was a negative connotation associated with the usage of PrEP as a prevention strategy. In social studies surrounding PrEP individuals did not want to participate in using the drug to avoid social stigma connecting drug adherence promiscuous, high-risk sexual behavior. Negative ideas around taking the medication in association with negative individual perception had a strong correlation when PrEP was first introduce. This was primary limitation in the effectiveness of PrEP as a strategy in combating the epidemic. If you cannot get individuals to take medication secondary to skepticism and conspiracy theory you are unable to establish a useful drug in combating the epidemic (NCBI – Eaton, Kalichman, Price, Finneran, Allen, Maksut. In 2017 the CDC released a statement reviewing the three studies that determined HIV positive partners that achieved an undetectable viral load status via suppression under ART therapy had negligible risk in transmitting HIV to negative partners during unprotected sex (CDC 2017). This statement was and continues to be monumental in the frontier of combating HIV including the social stigma surrounding persons tested positive with the disease. This step prompted national and international campaigns promoting U=U, Undetectable equals Untransmittable. (UNAIDS 2018). This step shows treatment as a form of practicing prevention. This has pushed health agencies and governments in targeting high risk individuals, locating positive individuals not on ART therapy and getting them care in order to suppress viral loads. Along with Truvada for Prep prevention of the spread of HIV can be maintained on two fronts. (1) By providing high-risk individuals tested negative for HIV with access and adherence to PrEP and (2) By finding HIV positive individuals and providing them with the adequate resources to establishing and maintaining viral suppression via ART therapy.   Bibliography(2018 – Demystifying Medicine) 2018 Demystifying Medicine: Why is HIV still a biologic and epidemiologic challenge worldwide)https://www.youtube.com/watch?v=pL3PUrqceAQ&t=5407s (Aids Info –NIH) https://aidsinfo.nih.gov/understanding-hiv-aids/fact-sheets/19/73/the-hiv-life-cycle(Aids Map) http://www.aidsmap.com/Viral-load/page/1327496/(Beta Blog HAART)https://betablog.org/haart-chart/(CDC 2017)https://www.cdc.gov/hiv/library/dcl/dcl/092717.html(CRISPR/Cas9)http://sites.tufts.edu/crispr/genome-editing/(FDA access data)https://www.accessdata.fda.gov/drugsatfda_docs/label/2006/021752s005lbl.pdf (Khan Academy – Diagnosing HIV)Concepts and tests – Infections diseases NCLEX-RN – Khan Academyhttps://www.youtube.com/watch?v=3CjCAeGhtHA (Khan Academy – How HIV kills) How HIV kills so many CD4 T cells – Infectious  diseases – NCLEX-RN – Khan Academyhttps://www.youtube.com/watch?v=8gnpnUFNloo (NCBI – Arrildt, Joseph, Swanstrom)https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3658113/(NCBI – Lopalco)https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3185609/(NCBI – Eaton, Kalichman, Price, Finneran, Allen, Maksut)https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5926187/(NIAID – Prevention) https://www.niaid.nih.gov/diseases-conditions/hiv-prevention(NIAID – Replication)https://www.niaid.nih.gov/diseases-conditions/hiv-replication-cycle (NIAID – Treatment) https://www.niaid.nih.gov/diseases-conditions/hiv-treatment(PBS – TruvadaPrEP)https://www.pbs.org/newshour/health/8-things-didnt-know-truvadaprep(Rx-List – Truvada)https://www.rxlist.com/truvada-drug.htm#description(TRACO 2015)https://www.youtube.com/watch?v=Kx7x0yofoUU(UNAIDS 2018)http://www.unaids.org/en/resources/presscentre/featurestories/2018/july/undetectable-untransmittable