HIV/AIDS exam #2
HIV Worldwide
- 1.8 million new cases of HIV in 2016. - 36.7 million people were living with HIV around the world in 2016, and - 19.5 million of them were receiving medicines to treat HIV, called antiretroviral therapy (ART). - An estimated 1 million people died from AIDS-related illnesses in 2016. - Sub-Saharan Africa, which bears the heaviest burden of HIV and AIDS worldwide, accounts for about 64% of all new HIV infections. - Other regions significantly affected by HIV and AIDS include Asia and the Pacific, Latin America and the Caribbean, and Eastern Europe and Central Asia.
starting ART early raises issues:
- Financial costs: ARV drugs are expensive - Adherence: the longer on ART, the more difficult it is for someone to respect the drug schedule - Drug resistance: The longer on ART, the higher the chance of developing drug resistance - ARV Drug-related toxicity (side effects) - Unknown long term risks of ART
Confidentiality vs. anonymity
- HIV testing may be confidential (test linked to individual's name; coded) - or anonymous (no link)
HIV in the US
- More than 1.1 million people in the US were living with HIV in 2016 - 1 in 7 of them don't know they are living with HIV - In 2016, 39,782 people were diagnosed with HIV in the U.S. - From 2008 to 2014, the estimated number of annual HIV infections in the U.S. declined 18%. - Gay and bisexual men, particularly young African American gay and bisexual men, are most affected, 2016
Known cases of HIV resistance in humans
- Steve Crohn (New York; 1946-2013) named "The Man Who Can't Catch AIDS" by The Independent newspaper (UK): gay NYC male with CCR5 △32 mutation - The Berlin Patient aka. Timothy Brown (Seattle, WA; 1966-): HIV+ male (since 1995) who underwent bone marrow transplant from CCR5△32 homozygous donor in 2007. Cured of HIV infection. - Non-progressors HIV+ children (South Africa, 2016)
Who Should be Tested for HIV??
- The CDC recommends that everyone between 13-64 years should be tested for HIV - HIV treatments are very efficient & can prolong lives - Knowing one's status may prevent further transmission (infection spreading) - Individuals in identified risk groups maintaining high risk of exposure behaviors should be tested more frequently (Every 6-12 months)
Reporting rules for HIV test results
- Vary by states: most U.S. states require medical personnel to report anyone with a positive HIV test to the State Health Department. - Ultimately, all data is reported to the CDC - Every person should have access to information related to reporting and how confidentiality is to be maintained
Blood serum's contents:
- proteins (including antibodies) - antigens/Ag - hormones - electrolytes - any foreign agent(s) (virus/bacteria etc...) that circulate in the blood. Plasma is ≠ than serum (blood treated with anticoagulant still contains clotting proteins but devoid of RBC & WBC).
Down side to Nucleic Acid Tests
- very technical (no home test version) - expensive (Cost of NATs is high: $60-$100) - require expensive equipment and highly trained laboratory laboratory staff - NATs cannot detect all HIV-1 subtypes equally, nor HIV-2
General pattern of progression of an HIV infection THREE phases:
1) Acute (10-12 weeks): - General symptoms (virus infection) high viral load reduces CD4-T cell count. - Cell count recovers & activates immune system 2) Chronic or latent (~ 8-10 yrs) - Almost asymptomatic - Lower viral load due to immune response which eventually fails to eradicate the virus (destruction of LNs) 3) Onset of AIDS (> 8-10 yrs) - Exhausted immune system fails (no more regeneration), virus load increases when threshold CD4-T cell count reached (~200 cell/mm3)
SIX Strategic Targets for Anti-HIV Therapies
1) FUSION/ ENTRY inhibitors (CD4- &/or CCR5-binding & fusion) 2) REVERSE TRANSCRIPTASE inhibitors - NRTI: Nucleoside Reverse Transcriptase Inhibitors - NNRTI: Non Nucleoside Reverse Transcriptase Inhibitors 3) INTEGRASE inhibitors 4) (Viral) TRANSCRIPTION inhibitors 5) Virus ASSEMBLY & PRODUCTION inhibitors 6) PROTEASE & MATURATION inhibitors
Five Generations of HIV Tests
1) First generation EIAs (ELISA Immuno Assays). - Used an HIV antigen extracted from virus (viral lysate) to detect any anti-HIV antibodies (IgG) - Window period: 56 days 2) Second generation recombinant HIV proteins or synthetic peptides - Detect anti-HIV-1 and anti-HIV-2 antibodies (HIV-1 IgG and HIV-2 IgG) - Window period: 42 days 3) Third generation Combination or "combi" tests: - Detect HIV-1 groups M & O, and HIV-2 - Use recombinant or synthetic peptides to detect anti-HIV antibodies (IgG and IgM antibodies) - Window period: 22 days 4) Fourth generation tests: (since 2000) - Detect HIV-1 group M and HIV-2 antibodies (IgG and IgM) and the HIV-1 p24 antigen - Window period: 15-17 days 5) Fifth generation tests: (as of 2014) Multiplex screening test that detects AND differentiates all three HIV biomarkers: HIV-1 antibodies, HIV-2 antibodies, and the HIV-1 P24 antigen
Early treatment benefits are 3-fold:
1) HIV+ individual's quality of life is improved 2) HIV+'s sexual partner is less at risk of infection 3) More cost effective as general health of individual is somehow "managed"
HIV testing has 3 purposes:
1) screening of blood supplies and donated organs 2) surveillance of HIV 3) diagnosis of infection. Anti HIV antibodies are much easier to detect than the virus itself and tests involving Ab are easier and quicker
A PCR cycle consists in 3 timed steps performed at different temperatures in a thermocycler:
1. Denaturation (95∘C): The dsDNA template becomes single stranded as helix's H-bonds are destroyed by heat. 2. Annealing (55-65∘C): Short complementary primers (Fwd & Rev) anneal to (=hydrogen bond with) their complementary target sequences in the newly ssDNA template. 3. Extension (72∘C): in vitro DNA replication occurs from each primer- "primer extension"- as each primer provide a free 3'OH end for the DNA polymerase to start complementary strand synthesis.
Advent of the Highly Active AntiRetroviral Therapy (HAART)
1981-1987- Dark age of the HIV/AIDS epidemic: Thousands of HIV infected people suffered horridly and died of AIDS globally due to the lack of effective anti HIV treatment. 1987- AZT/Zidovudine: NRTI- abandoned cancer drug- proved effective to slow the progression to AIDS in HIV+ individuals 1991: a SECOND antiretroviral drug is available - didanosine DDI (RT inhibitor) 1995 FIRST protease inhibitor is marketed & available for treatment of HIV infections 1996- : Beginning of a new era for HIV+ individuals with the availability of the Highly Active Antiretroviral Therapy (HAART) - Pre HAART era (1981-1996= 15 years) No effective HIV treatment - Post HAART era (1996- today): Effective HIV treatment
Testing for HIV is available since ______
1985
Western Blot Analysis steps:
1st step: Electrophoretic separation of serum proteins (containing HIV proteins & anti HIV Abs) by size in a polyacrylamide gel matrix (NOT agarose gel): SDS-PAGE analysis - Sodium Dodecyl Sulfate (SDS) detergent, (-) charged, coats all unfolded protein negatively - Migration of (-) charged proteins from - (cathode) to + (anode) in gel. - Addition of ß-mercaptoethanol in sample buffer, & heating of protein sample (95∘C/5 mins) prior to loading breaks up disulfide bridges (S-S) & H-bonds in proteins (respectively): proteins denature & unfold into linear peptides that will migrate by SIZE (from top of gel/well to bottom). 2nd step: transfer of gel content i.e Western-blot (proteins in their migration profile) onto a PVDF membrane -Polyvinylidene fluoride; solid membrane "sticky" for proteins- for immunodetection using an Ab directed against the target protein in the WB (HIV Ag or anti-HIV Abs). - Contents of SDS-PAGE gel (proteins in their position of migration/ by size) are electro-transferred onto a solid membrane (nylon or other polymer/PVDF). - Immunodetection is performed using 1ary & 2dary Abs on the solid WB membrane - membrane is a replica of gel that holds all transferred serum proteins.
The 1st Human Preventative HIV Vaccine Trial
2009 Preventative HIV Trial in Thailand conducted by a U.S. Army HIV/AIDS Research Program Two stage "prime-boost" RV144 vaccine tested on 16,400 HIV negative males & females (18-30 yrs of age) reduced the risk of HIV infections risk by ~30% - RV144 combined 2 vaccines inefficient on their own: the prime ALVAC HIV and the boost AIDSVAX B/E - Hypothesis: RV144 may induce different immune responses that may enhance overall immune response to HIV antigens & provide protection. The vaccine failed to reduce viral load in some subjects who became infected with HIV during the trial - RV144 was not designed to protect against ALL HIV strains - RV144's efficiency dropped over the 1st year of trial= temporary protection against HIV was provided (None of the HIV infections in subjects were induced by vaccine itself)
The Mississippi Baby (2013)
2010-Baby girl born HIV+ (mother-to-child transmission) - Mother DID NOT receive ARV treatment during pregnancy - Baby treated very early (30h after birth) with aggressive ARV treatment - ART interrupted due to missed appointments (at 18 month old) HIV remission- temporary- Despite 5 months of ART interruption, the toddler kept her viral load in check for 27 months - Blood tests showed no virus replication though provirus presence was confirmed (PCR). 2014 (July)- Viral rebound: Baby who was thought to be functionally cured eventually became HIV seropositive - Blood tests indicated Viral Load of 10,000 HIV copies/mL - ART decreased VL to undetectable levels (<50 copies/mL) - HIV strain was 99% identical to mother's which means that latent HIV reservoirs were created early meaning baby was infected during gestation, not vaginal delivery Since then, other babies ("Milan Baby" & "Canadian Baby") with similar aggressive early ARV treatment showed different timing for HIV remission and viral rebound - Transient remission times differ depending on the numbers of existing HIV reservoirs & the timing of the immune response activation - The ability to suppress viral load in a short time correlates with lower reservoir size - immune activation varies between individuals. Early aggressive ART strategy in infants is efficient to reduce the amount of HIV reservoirs created and increase remission times (event if HIV remission is transient)
Using Gene Therapy for HIV
2014- 1st time gene therapy is relatively safe and efficient to genetically modify cells to be resistant to HIV infection (humans) ZFNs target membrane CCR5 co-Receptor in CD4+ T-cells Genetically modified CD4+ T-cells (deletion of CCR5 allele) were infused in 12 patients - Aeviremic patients (undetectable viral load) on ART GM T-cells once engrafted survived 48 days in all patients - 1 patient had adverse treatment reaction to transplant Viraemia analysis post-ART treatment interruption showed better survival for GM T-cells compared to non-GM T-cells. - HIV undetectable in 1 patient (heterozygote for CCR5 allele deletion i.e. single allele copy) after ART interruption - Therapy is efficient in patients left with a single copy of the CCR5 allele in their genome
There are ____ current NNRTIs
4
There are ____ current NRTIs
7
Complete Cure for HIV infections?
A functional cure for HIV/AIDS MAY BE possible & allow HIV+ individuals a basically asymptomatic life and cancel disease progression to live without progressing to AIDS Very few examples of functionally cured people exist: - The Berlin Patient (as of 2006) & the Mississippi Baby* (2013) (*The Mississippi Baby tested HIV+ in 2014)
South Africa's HIV+ non-progressor children
A rare group of children are resistant to the effects of HIV in South Africa Non-progressors HIV+ chilren: High viral load in blood, yet stay healthy. Characterized by very low levels of immune activation (HIV fails to activate their immune system). The immune system of all non-progressor children is NOT destroyed by HIV (= NO AIDS onset).
State of HIV Vaccine Development
A total of 4 vaccine trials since the 1st human trial in 2009: 1) 2009 RV144 2) 2013 HVTNO97 3) 2015 HVNT100 4) 2016 HVTN702 Each time, vaccine efficiency of the vaccines improved (slightly) as bioengineered vaccines are more refined (better design)
What is a Vaccine?
A vaccine is composed of antigens (parts of a pathogen) or the entire (neutralized/disarmed) pathogens - The vaccine is injected in the blood to illicit an immune reaction. Antigens trigger the Antibody-Mediated Immunity path: - Production of antibodies (Ab) against the antigens (Ag) - Establishment of a record or immune memory of the infection to respond faster to any future infections by this Ag/pathogen. Researchers may take many years to develop a specific vaccine - Vaccines are expensive to develop and produce
Principle of the ELISA Test Direct Assay and Indirect Assay:
An HIV antigen (produced recombinantly- viral gene expression & viral protein/Ag purification) may coat the microtiter plate's wells (or bind to lose beads: see Fig. 7.01 Weeks Textbook). If any anti-HIV antibodies (human anti-HIV Ab are immunoglobulin G, IgGs) are present in the patient's serum (blood), these will bind to the recombinant HIV Ag. The anti-HIV Ab/HIV Ag complexes are detected with another enzyme-conjugated horse anti-human IgG antibodies - Anti-human IgG binds to human IgG (=anti HIV Ab).
Point-of-Contact (Rapid) 4th Generation HIV Testing
Alere Determine HIV-1/2 Ag/Ab Combo - 4th generation for fingerstick or venous whole blood, serum or plasma - Can be used to detect acute (early) HIV infection before antibody detection - Distinguish between the detection of p24 antigen and HIV antibodies - Results in about 20 minutes
Types of HIV Tests Available
Anti-HIV antibody tests: Only detect the anti HIV- antibodies produced by the individual post HIV infection - Anti-HIV antibodies present in blood (serum). Combined HIV antigen-anti HIV antibody tests: Detect the presence of the virus (viral antigens) AND the presence of anti HIV antibodies produced. - Best to identify early HIV infection compared to anti-HIV antibody only tests HIV RNA tests: Quantitative tests performed on blood measure the amount of HIV RNA i.e. the viral load
Nucleic Acid Tests (NATs): Viral Gene Tests
Assess directly the presence of the virus through the amplification of viral gene sequences (direct tests) from the virus/provirus' genome (integrated DNA or free RNA). - Rely on the use of the Polymerase Chain Reaction technology (PCR) PCR is a very specific in vitro DNA replication process that allows to make several copies of a target DNA sequence (i.e. viral gene of interest) between borders present in small quantity in a sample (DNA fragment amplification). - PCR relies on the use of a high fidelity heat resistant DNA polymerase - DNA target amplified between two complementary primers (opposite sense) RT-PCR (reverse Transcription PCR) is a modified PCR technique that targets a specific part of an RNA template & reverse transcribes it into a complementary dsDNA (cDNA) sequence that accumulates in the sample. - The enzyme used in RT-PCR is a REVERSE TRANSCRIPTASE (uses RNA as a template to synthesize complementary dsDNA (just like a retrovirus) PCR & RT-PCR detect the viral nucleic acids (DNA with PCR, or RNA with RT PCR), hence they both detect HIV itself - PCR & RT-PCR do NOT detect gene products i.e. proteins: anti-HIV antibodies/Ag. PCR and RT-PCR are qualitative & quantitative techniques that can determine the number of viruses in a patient i.e. the viral load (or viral burden). - DNA is more stable than RNA. - PCR is easier (DNA template) than RT-PCR (RNA template; degradation) Knowing the amount of HIV particles in a patient helps understand the relationship between viral load and HIV/AIDS and monitor/assess the efficacy of anti-viral drugs.
Disadvantages to Using Bacteria
Bacteria are unable to process and correctly modify many eukaryotic proteins - Cannot add carbohydrates (glycosylated proteins) or phosphorylate like eukaryotic organisms Solution - many biopharmaceuticals are now produced in eukaryotic hosts: - Bioreactors or biofactories: Living factories, such as a herd of goats or cows - Baculovirus: Gene delivery system in which a virus is used to infect insect cells
The Latest Preventative HIV Vaccine Trial
Based on 2009 Thailand human trial (& later 2013 & 2015 trials), a NEW HIV vaccine human trial started in South Africa in Nov. 2016: HVTN702 HIV Vaccine Trial will test efficiency of "ALVAC-HIV (vCP2438) + Bivalent Subtype C gp120/MF59" 5400 subjects (HIV- men & women; 18-35 yrs old) Vaccine does NOT contain the live virus - Contains bio-engineered pieces of the virus, causing an immune response in the body - Vaccine targets HIV-1 subtype C (prevalent form in Southern Africa). Vaccination requires 5 injections within a year & patient monitoring for the next few years End of trial: July 2021 (Currently in phase III) - Target: 50% vaccine efficiency will trigger vaccine licensing & manufacture
Synthetic Insulin
Bioengineering process of insulin synthesis 2 insulin subunits are produced as a fusion protein - Fusion proteins: Hybrid protein (i.e. "chimeric") Fusion protein purified cleaved to release insulin polypeptides 2 subunits spontaneously unite and form active insulin
Biopharming
Biopharming - Production of proteins of interest in genetically modified plants and animals (After cloned gene delivery to GMO) Biopharmaceutical products - Recombinant proteins - Therapeutic proteins that treat disease
Integrase inhibitors (Integrase Strand Transfer Inhibitors)
Block HIV integrase's activity thereby preventing the integration of the viral cDNA into the host cell's chromosomes - 2007- FIRST integrase inhibitor approved by FDA: Raltegravir (Isentress; MK 0158; Merck): $16,092/yr/person - 2013: Dolutegravir (Tivicay) $17,040 - A NEW integrase inhibitor which is still in late clinical development by Gilead Science shows great promises: Elvitegravir - Elvitegravir reduced viral load to 50 copies/ml in experienced patients with extensive drug resistance - More efficient than comparative protease inhibitor trial results - Elvitegravir seems to be well tolerated (very few participants stopped taking the drug due to side-effects) - ANTICIPATED ISSUE: Elvitegravir may interfere with another integrase inhibiting drug Raltegravir (Isentress)
what mode of reporting is most commonly used in the US?
Confidential mode is most common as some US states' laws require that the number of HIV+ individuals be reported (no names used)
DNA-Based Vaccines
DNA (=gene) encoding proteins from pathogen inserted into a vector Recombinant plasmid is injected into individual Production (gene expression) of pathogen protein elicit an immune response (and immune memory). Triggers immune response if individual becomes exposed to the real pathogen in future Trials are underway using plasmid DNA encoding protein antigens from HIV in an attempt to vaccinate against HIV
CDC PrEP Guidance: Who is recommended for PrEP?
Daily oral PrEP is recommended for adults at substantial risk of acquiring HIV infection: - Sexually active MSM - Heterosexually active men and women - Injection drug users
Timing of HIV or HIV Antibody Detection
Depending on the person, once infected with HIV, the immune system may take up to 3 months to produce any Ab against HIV. - The seroconversion window period is the time between HIV infection & anti HIV antibody production by the infected individual. (person will test HIV- during this period) A seronegative result or HIV- (= no detectable anti-HIV Ab in serum) does NOT mean the person is NOT infected with HIV if the test occurs anytime during the first 3-6 months after infection. - Once anti-HIV antibodies are produced (& detected), the infected person has seroconverted = he/she now tests HIV+ (HIV seropositive). The proper test (anti-HIV Ab detection vs. Nucleic Acid Tests) must be performed when the target molecule (Abs) or agent (through HIV RNA/cDNA or HIV Ag) are present. Viremia refers to the presence of virus in the blood
Different Types of ELISA Tests
Direct assay - Use of a single Ab conjugated/linked to assayable enzyme E (colorimetric or chemiluminescent reaction). - This E-conjugated Ab binds to the viral antigen Ag coating the microtiter plate' well: Color = virus presence. Indirect assay - A Primary antibody binds to the immobilized viral Ag. A secondary Ab (conjugated to an assayable E) binds to the primary Ab & allows for the detection of the complex: Color = virus presence. Capture assay ("sandwich') - Capture anti-HIV Ab fixed in plate's well binds to viral Ag. primary Ab (same as capture Ab) binds to the viral antigen & complex is detected through secondary Ab conjugated to E. Color = virus presence. - This technique is the most used as secondary HIV test
Mutant HIV resistant strains can be identified through different tests:
Genotypic testing: - Cheap ($200-400) and quick(1-2 weeks) - Viral gene or genome sequencing to identify known mutations that confer viral resistance to specific drugs Phenotypic testing : - Easy but costly (800-1000$/test) and long (2-5 weeks) - Estimates rate of HIV reproduction under drug pressure in vitro
Protease inhibitors
Disable HIV's protease to prevent HIV virion maturation - Only mature virions are infectious - Protease inhibitors result in immature virion production (Stop further infection by new virions) - PR inhibitors are efficient as they do NOT affect human proteases at all (limit side effects/toxicity). PIs are very potent but may may be dangerous because they may interfere with other drugs (in some case, interference is lethal). Common side effects: Liver toxicity, hypoglycemia, flatulence, bloating, lipodystrophy (fat distribution) Seven new protease inhibitors are in testing phase currently in the U.S. As of 2014, 16 HIV protease inhibitors were under investigation: 6 were in clinical trial phases, and 10 PIs were approved by the FDA 28 other ARV drugs are also being investigated.
Antiviral and Antiretroviral Drugs Fight Viral Infections
Drugs that fight viral infection MUST prevent or reduce viral replication (the production of virus progeny) Antiviral medications were developed by targeting various events of the viral replication cycle Chemical compounds that target retroviruses like HIV are called antiretroviral drugs (ARV) - Different ARV drugs target different mechanisms or steps of the HIV life cycle Goal of antiretroviral therapy or ART is to reduce viral replication in HIV+ to undetectable clinical assay levels - HIV-RNA< 50 copies/mL
Accuracy of the ELISA Tests
ELISA tests are 99.9% accurate (but tests are not infallible) - 0.1% (1/1000) chance to diagnose HIV+ when HIV- (usually human error) - ELISA is NOT the most accurate test for HIV, BUT many samples can be screened at a relatively small cost very quickly.
The seroconversion window period consists in:
Eclipse period: Right after infection, (lasts ~1 month= start of window period- none of the current HIV tests can evaluate the infection status - No to little viremia (under detection limit) & no immune response yet As viremia occurs, the HIV+ subject becomes infectious (high risk of infection through bodily fluids) & experiences HIV infection symptoms ~ 11 days into the infectious window period is the acute infection stage (RNA peak), NATs -Nucleic Acid Tests- & immuno-assays of HIV Ag can detect the presence of HIV in the blood. Immune system (IS) then kicks in & produced anti-HIV Abs are detectable. - IS fights off the infection, consequently, the blood's viral load drops down: symptom-free period. - When viral load increases again (p24 & RNA levels rise), status evolves to last stage of infection i.e. AIDS.
HIV's Life Cycle
Entry: Binding of HIV to T-cell Receptors & co-R, & fusion of viral & host membranes Release of viral RNA genome & reverse transcription of into cDNA (RT enzyme) Integration of viral ds cDNA into host's DNA Viral gene expression: transcription & translation Virion assembly, maturation (proteases) & release (budding)
HIV Subunit Target Proteins
Envelope (env) glycoproteins gp120 and gp 41 and their gp 160 precursor are ALL immunogenic Capsid protein p17 - Does NOT mutate as rapidly as env proteins (Less mutations in gag genes mutate compared to env genes) However, subunits like gp120, gp41, and p17 are NOT HIGHLY immunogenic - do not trigger a large production of antibodies These proteins are not easily engulfed by macrophages or efficiently delivered to the immune system This problem is oftentimes solved by chemically binding of these proteins to "carrier" molecules aka adjuvants (aluminum sulfate, liposomes) Adjuvants are known to increase immune response Antibodies prevent binding of HIV to host cells. - HIV adsorbs to a host cell with gp120 protein which binds to CD4 on the host cell. After adsorption, gp41 stimulates fusion and penetration. Antibodies against gp120 will bind to gp120 and prevent gp120 from binding with CD4. Antibodies against gp41 will inhibit fusion and entry of HIV into cell.
Difficult Access to HIV Conserved Env Ag Epitopes
Epitope: Specific part of the antigen (Ag) recognized by its cognate antibody (Ab- produced against said Ag) and allows for Ag-Ab binding. Glycans (carbohydrates=sugars) linked to HIV glycoproteins mask critical antigenic sites. GP120 & GP41 also change conformation & camouflage the highly conserved CD4 binding sites (aa sequence) - Trimer formation at surface of HIV Targeting highly conserved CD4-binding sites in GP120 & GP41 is essential to develop a vaccine that is efficient against ALL HIV groups, subtypes, & strains. - Same CD4-binding sequences exist in ENV proteins across HIV types, groups and strains - Mutations in CD4 binding sites have NO selective advantage for HIV: Mutations are NOT selected in new strains as CD4-binding function is essential to HIV entry for its replication
FTC/TDF (TRUVADA) FOR PrEP INDICATION
FTC/TDF is indicated in combination with safer sex practices for PrEP to reduce the risk of sexually acquired HIV-1 in adults at high risk This indication is based on clinical trials in MSM at high risk for HIV-1 infection and in heterosexual serodiscordant couples
Criminalization of Non-Disclosure HIV May Have Negative Consequences
For example: Imprisoning people living with HIV may not prevent HIV transmission. - prisons are environments in which high-risk behaviors are common. HIV prevention measures are limited. - This may lead to the transmission of HIV within prisons and then more broadly when people in prison return to their communities. Prevention interventions, such as education and risk-reduction counselling, may be better suited than criminalization for changing complex human behaviors related to sex and drug use. The fear of criminal prosecution may deter people from being tested. If someone doesn't test for HIV, then they won't know their HIV status and thus may believe that disclosure and/or onward transmission of HIV isn't an issue for them. Extensive use of criminal prosecution could lead to a misperception by the public about the risk of transmission. - This is especially relevant in cases where stiff sentences are imposed for behaviors associated with a negligible risk of transmission. The public attention given to criminal prosecutions may create a false sense of security that the law will protect people from HIV infection. Criminal prosecution can add to the stigma and discrimination faced by people with HIV. - It places the burden of preventing HIV transmission on those living with HIV and portrays those living with HIV as potential criminals. Confidentiality of records can be breached when evidence is being gathered for a prosecution and the identity of the person living with HIV is revealed. - This can lead to stigma and discrimination. It can also undermine trust in the healthcare system. Gender and power inequity can make the situation more complex. - For example, some HIV-positive women may not be able to insist on condom use or may fear violence if they reveal their HIV status.
HIV/AIDS: Functional vs. Complete Cure
Functional cure: Aggressive ART early on to limit & reduce viral load to undetectable levels & strengthen the immune system to keep the disease under control when ART is discontinued. - Unlikely transmission as viral load is too low. - Disabled HIV though provirus still present. Complete cure: Complete elimination of the virus particles, and any trace of the the provirus (integrated) genome from all the cells. - Cured patient does not need ART anymore. The main issue in finding a complete cure is the existence of persisting latent HIV reservoirs
Targets for HIV Antiretroviral Drugs
GP120 viral envelope protein interaction with CD4 receptor on T-cells release of viral RNA from the capsid Viral reverse transcriptase enzyme Viral genome (2 ssRNA+) Viral particle assembly Virion release through budding
Genetic Engineering
Genetic engineering - Alteration of organism's genome using recombinant DNA technologies to add or remove gene from genome - Produces genetically modified organisms (GMOs) Biotechnology - Utilizing living organisms to create a product or process that helps improve life for humans or other organisms
HIV Subunit Vaccines
Genetic engineering techniques allow for the production of viral subunits in mass quantities: Identification & isolation of the gene coding for viral protein (Ag) Incorporation of a viral gene into bacterial plasmid Recombinant plasmid inserted in a bacterium Ag/Protein of interest expressed Ag/Protein of interest harvested and purified Fragments mimicking entire organism, contain only essential Ag. The in lab grown virus is broken apart chemically to separate important antigens for use in vaccine development. Viral Ag mimics organism's presence and stimulates the immune response. This is a very safe approach as a viral subunit alone CANNOT infect any cells (no genetic material). After infection, Abs bind the target antigenic subunit portion in the live virus or bacteria to neutralize it.
Genome Editing Technologies
Genome editing is possible through specific types of technologies developed from known DNA-binding proteins & DNA cutting enzymes (nucleases) These genome editing tools are molecular scissors that have "surgical precision" because they can be engineered to bind to any DNA sequence's location - Break the dsDNA or chromosomes at wanted (user-specific; engineerable) locations in the genome & can: ~ Mutate (insertion or deletion) a defective gene ~ Precisely insert a donor gene of interest to replace a defective gene (The joining of DNA fragments (insertion) occurs through natural processes of recombination (DNA repair of double stranded breaks): Homologous vs. non homologous recombination.) Zinc Finger Nucleases (ZFNs) Transcription Activator-Like Effector Nucleases (TALENs or TALE Nucleases) Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR Associated protein 9 or CRISPR-Cas9 - RNA-guided gene editing tool: MORE precise, EASIER to use/engineer, and cheaper than ZFNs & TALENs. - Cas9 = restriction nuclease. - Uses a synthetic guide RNA (gRNA) to introduce DSB (double stranded breaks) in DNA Once engineered, the ZFN, TALEN, or CRISPR-Cas9 constructs are cloned into a plasmid that is delivered to the host cells to be genetically modified/edited.
RNAi targets for inhibition of HIV-1 replication
HIV enters the cell by binding to cellular receptor (CD4) and co-receptor (CCR5 or CXCR4). Following entry and uncoating, the viral RNA genome is reversely transcribed and the proviral DNA integrated into the host's genome. Proviral transcription generates viral mRNA that encodes viral proteins (gag, pol, env, etc...) Any region in the viral mRNA and/or mRNA for host genes involved in HIV-1 life cycle- like CCR5- can serve as viable RNAi targets to suppress viral replication.
Which HIV proteins are detected?
HIV proteins are the target of some HIV tests. Using protein-based serological HIV tests, the presence HIV may be detected directly (P24 protein antigen) or indirectly through any human antibodies raised against the different ENV or GAG viral proteins - ENV: GP120, GP41, GP160 precursor -GP160 is cleaved into GP120 & GP41 (protease). - GAG: P24/capsid
Issues Preventing a Complete Cure for HIV infections
HIV viral load elimination in blood & destruction of infected T-cells actively producing HIV particles WILL NOT cure HIV infections - Existence of latent HIV reservoirs HIV lurks-resting state-as provirus present in: - Semi-quiescent memory T-cells - Active T-cells if inserted in a transcriptionally silenced region of the genome (epigenetic position effect). ART cannot eliminate inactive viruses All latent HIV reservoirs must be ALL destroyed to completely cure an HIV infection - Latent reservoirs must activated to be destroyed (Activation of proviral genome expression leads to detectable HIV presence (viral proteins & virion production))
Importance of Viral Load in HIV/AIDS Treatment
HIV viral load is the most important indicator of response to ARV therapy No HIV viral load level is completely safe Undetectable vs suppressed viral loads - Undetectable viral load: Risk of HIV transmission when viral load falls <50 copies/ml is extremely low. (but NOT ZERO) - Undetectable viral load is the target of ARV therapy - Notion of undetectable viral load changes constantly as techniques to detect virus evolve and become more sensitive. - An undetectable viral load does NOT mean that there is NO HIV in the blood... (often HIV is in blood, lymphoid tissues and bodily fluids eventhough viral load is undetectable) - Suppressed viral load: HIV particle number/ml blood < 400 In patients with undetectable viral loads, clinically significant viral evolution comes to a halt, and CD4+ cell count increases - Immune system is back online - HIV infection is NOT cured (existence of LATENT HIV reservoirs inaccessible to ARV drugs)
Treatment as Prevention:
HPTN 052-96% Reduction in HIV Transmission
Problems with HIV Vaccine Strategies
High virus mutation rate Destruction of T-lymphocytes Danger of inactivated HIV virus-safety precautions - Reports of non-pathogenic viruses converting to the pathogenic form Neutralizing antibodies were found to keep the virus "in check" for a very long time (from HIV+ individuals) Hope to develop vaccine which would trigger the immune response (capsid protein, envelope proteins, reverse transcriptase)
Timothy Brown aka the Berlin Patient
In 2007, American Tim Brown, HIV+ since 1995, underwent acute myeloid leukemia (AML) treatment in Germany. - Chemotherapy & radiation annihilated Brown's immune system completely (elimination of latent HIV reservoirs) - His doctor, Gero Heutter, rebuilt Brown's immune system using a compatible rare genetic donor who was resistant to HIV infections for a bone marrow transplant (Donor carried the CCR5△32 homozygous gene mutation: HIV cannot enter T-cells that carry the mutant CCR5 (R5)) Brown's body accepted the transplant and became "HIV-" - no ART treatment needed & still has (2017) an undetectable viral/HIV load Tim Brown is a unique example of functional HIV/AIDS cure: - Costly experimental scientific procedure: 100s thousands of dollars; 1 year of convalescence in hospital; - Difficult to replicate & inaccessible by most HIV+ individuals - Not all HIV strains enter T-cells through CCR5 receptor. - Harsh chemo treatment almost killed Tim Brown
Starting Antiretroviral Therapy (ART) in HIV+
In the U.S. (NY & SF) guidelines recommend to start ART therapy as soon as HIV+ status is known regardless of CD4+ cell count. starting ART early raises issues: - Financial costs: ARV drugs are expensive - Adherence: the longer on ART, the more difficult it is for someone to respect the drug schedule - Drug resistance: The longer on ART, the higher the chance of developing drug resistance - ARV Drug-related toxicity (side effects) - Unknown long term risks of ART
Inactivated vs. Attenuated Virus Vaccines
Inactivated virus vaccine - Viral particles are grown (lab) but unable to multiply in the body because of physical (heat) or chemical (formaldehyde) treatment, (or even radiation): safer option, inactivated viruses are stored in freeze-dried containers (polio) - Inactivated virus: inactive virus produced by chemical or physical treatment Attenuated virus vaccine - Weakened version of the virus which is still able to replicate in the body but at a such low rate that there is no disease onset. Closer to a natural infection. - Vaccine must be stored a 4 deg. C i.e. fridge. Ex. measles, mumps, and chickenpox - Attenuated virus: development of variants with reduced virulence
Potential of a Therapeutic HIV Vaccine
Increase life expectancy in HIV+ individuals Promote a disease-free period (symptom delay) Prevent the progression of the disease Virus elimination is unlikely because of latent HIV reservoirs Reduce the viral load in HIV+ - Decrease the probability of HIV spreading - Decrease the probability of transmission to offspring
Risks associated with the HIV vaccine
Increased risky sexual behavior Resistant virus escape and transmission Uncertainties - Actual viral load reduction - Disease progression - Duration of the vaccine effect - Time between vaccinations - Increase in risk behavior - Escape of the resistant virus
Insulin Production in Bacteria
Insulin produced by pancreas regulates blood sugar levels Insulin production in bacteria - Therapeutic proteins can be produced by introducing human genes into bacteria Humulin - Synthetic or recombinant human insulin which was originally produced in bacteria (since 1982)
NEW DRUG CLASSES- NOT YET APPROVED BY FDA
Maturation inhibitors: Bevirimat & Vivecon Transcription inhibitors: Target the functions of viral Tat ((or Rev)) proteins - Phenylcoumarins - Antisense drugs: RNA interference/RNAi (HGTV43) prevent viral function ~Proviral genome in infected T cell is expressed and the viral RNA transcript is translated into viral polyproteins for viral components. ~An antisense RNA molecule with a complementary sequence of nucleotides to that of the viral RNA binds to it (the sense molecule) and prevents the translation of the viral RNA molecule (no viral protein production= no HIV virion can be produced!)
Enzyme-Linked Immunosorbent Assay (ELISA):
Quantitative & qualitative biochemical assay that detects antibodies directed against HIV antigen - Targets= proteins, peptides: viral Ag or anti-viral Ab (Ab & Ag are proteins). - Efficient technique for large scale screening (high number of samples
Development of Live vector Vaccines
Mild version of the -attenuated- virus (adenovirus, which often has several genes removed to reduce its virulence) recombined with the genetic material of a virulent virus (HIV for example). A vaccinated individual would develop the immune response to the viral protein produced by the (viral) Ag-encoding gene included in the recombinant (~hybrid) virus. Genes encoding key HIV proteins are incorporated into the genome of another virus (for example, adenovirus) Vaccinated individuals respond to the expressed viral proteins - This immune response includes antibodies targeting HIV Ag/proteins
Drugs that Target Adsorption
Mode of action of soluble CD4. - An HIV particle unites with a susceptible host cell when its GP120 molecules bind with CD4 molecules at the host cell surface. - Genetically engineered soluble CD4 molecules bind to a virus and prevent it from binding with the natural CD4 molecules. - Unable to bind to the host cell, the virus is rendered harmless.
Issues with NRTIs:
Monotherapy (single drug use) does not stop the HIV-RT 100% - When used individually they do not delay of AIDS onset - New drugs are mixes or fixed dose combinations of 2 or more drugs Short term efficiency of any given drug (resistance issue) Each drug has its own toxicity/side effects Costs for NRTIs like any ARV drugs is HIGH ($/per year per person) NRTIs like AZT (ZDV; Retrovir), d4T (Stavudine; Zerit), 3TC (Lamivudine; Epivir) can penetrate the blood brain barrier NRTIs' common side effects: Lactic acidosis
Reverse Transcriptase Inhibitors:
Nucleoside Reverse Transcriptase Inhibitors (NRTI): Azidothymidine/AZT (Zidovudine) - These are base analogs (lack 3'OH on sugar) that inhibit the RT enzyme by competing with regular nucleotides (dNTPs) - Incorporation of AZT in the nascent viral DNA strand stops synthesis - During reverse transcription, the RT enzyme needs a 3'OH (green arrow) from the last incorporated nucleotide to add the next complementary free nucleotide Used alone, NRTIs lead to HIV resistance but very efficient when used in combination with other ARV drug classes
Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTI): "non nukes"
NNRTI drugs bind to the HIV reverse transcriptase enzyme to inhibit its activity: - Stop viral DNA synthesis by inactivating RT directly - Different mode of action than NRTIs NNRTI have a mixed ability to penetrate the blood brain barrier. NNRTIs' common side effects: mild rash & liver damage When used alone, HIV rapidly develops resistance to an NNRTI drug - Mutant HIV strain with mutant RT enzyme that can no longer bind to NNRTI drug develop within weeks of NNRTI use Primary benefit of NNRTI is to delay protease inhibitor use NNRTI used effectively in triple therapy regimens Currently licensed NNRTIs: - FV efavirenz (Sustiva/Stocrin) - ETR etravirine (Intelence) - NVP nevirapine (Viramune) - DLV delavirdine (Rescriptor, only licensed for use in the US)
HIV Develops Resistance to Antiretrovirals
Over time, drug resistance may emerge in a population of HIV particles due to the high rate of mutations (reverse transcription errors; RT). Anti-HIV drugs like nevirapine - a non-nucleoside reverse transcriptase inhibitor/NNRTI of HIV-1- neutralize susceptible HIV particles. Some resistant i.e. mutant HIV strains multiply and become the dominant members of the viral population. At that point, the main strain of HIV present (mutant) will be resistant to nevirapine which will no longer useful. This newly acquired drug resistance may give rise to mutant superstrains of HIV - 2004 (NY) case: ~Gay male who had multiple partners (100s) & methamphetamine user ~Developed HIV-1 superstrain resistant to 19 of the 20 ARVs ~Succumbed to extremely accelerated progression to AIDS (1yr) Shift from single drug therapy to multiple ARV therapy in 1996: Highly Active AntiRetroviral Therapy (HAART) - Decreases the emergence of ARV resistance in HIV-1 strains - It is a race between the pharmaceutical industry and the highly and fast evolving HIV: ~New drugs from each different class, and different combinations of ARVDs, must be developed as the human HIV+ population as a whole breeds HIV resistance to the current drugs and drug combinations ~Combined drug therapy (different drugs from different classes) is MORE efficient to combat HIV infections than monotherapy.
Polymerase Chain Reaction-Steps
PCR cycle (~25-35x) 1. Denaturation of dsDNA template. 2. Annealing/hybridization primers-target ssDNA; dNTPs & Taq DNA Poymerase for rxn). 3. Extension= DNA replication btwn primer borders. Taq DNA Pol enZ - Newly synthesized DNA strands serve as template in next cycles. - Ultimately, the region amplified lies within the 2 primers. - Complementary short ssDNA PRIMERS (not called "gene probes") flank the target DNA sequence to be amplified by PCR
Parallel- & fast-track procedures for testing an experimental drug
Parallel-track procedure: experimental drugs are made available to certain patients during Phase I trials Fast-track procedure: Release of IND during Phase II trial while the trial is still ongoing. - A life-threatening illness could trigger a fast-track release.
Plasmid DNA Vaccines
Plasmid DNA vaccines consist in specific viral protein encoding genes recombined into non infectious plasmids. - Once the plasmid DNA vaccine is injected into patient, viral Ag genes on the plasmid are expressed to produce antigenic proteins that stimulate an immune response The modified-recombinant-plasmids carry a gene isolated from the virus which code for a particular antigen. - Plasmids are circular DNA fragments (from bacteria), are widely used in genetic engineering, and can be modified to be compatible with expression in human cells. These modified or recombinant plasmids are injected into the human muscle tissue and the synthetic protein production is stimulated. The immune response to the antigens is stimulated and the antigens are recognized as foreign. These types of vaccines are difficult to develop and pose ontogenesis concerns. DNA vaccine used to develop West Nile Virus vaccine. 2014-Experimental research in macaques show that these types of DNA vaccines efficiently induce humoral response
Cloning Genes into Plasmid Vectors
Plasmid vectors - Plasmids: circular DNA molecules from bacteria - Insert foreign DNA into plasmid using restriction enzymes (~ molecular scissors) - Linker in Multicloning Site: synthetic DNA fragments containing restriction sites - DNA Ligase enzyme serves as molecular glue to join the different DNA fragments
Vaccines: Preventative vs. Therapeutic
Preventative or Prophylactic vaccines: - Contain parts of the pathogen (antigen/Ag), or the disarmed pathogen (non infectious) - Administered to a healthy person who is not infected by the agent/pathogen for which they are being vaccinated for (immunization). - Goal: To elicit an immune response (antibody production) in order to build an immune memory of the pathogen for rapid immune response in the event of a future infection by this pathogen. - There are currently NO FDA-approved preventative vaccine against HIV/AIDS Therapeutic or treatment vaccines: Administered to a person who is already infected by the pathogen/agent for which the vaccine was designed against. - Goal: To treat the condition by strengthening the immune system of the infected individual to fight off the infection/condition (Form of immunotherapy) Examples of therapeutic vaccines: - 2010- FDA approved 1st therapeutic vaccine to treat some men with metastatic prostate cancer (Provenge®). - 2015- FDA approved 1st oncolytic virus therapy (T-VEC or Imlygic®) for treatment of metastatic melanoma - There are NO FDA-approved therapeutic vaccine against HIV/AIDS - Neutralizing Ab-based therapeutic vaccines would NOT cure HIV completely though they would reduce viral load & stop further T-cell infection in HIV+ individuals (infectious HIV particles would be neutralized) Functional vs Complete HIV/AIDS cure
Viruses are obligate intracellular parasites that use host cell machinery to:
Provide energy and molecule building blocks (amino acids, nucleotides) Provide replication machinery Synthesize viral proteins Provide envelope lipids Viruses HIJACK their host' s cell machinery to reproduce actively - A virus only contains its genome (nucleic acid) & key enzymes necessary for its life cycle (RT, IN, PR)
Western Blot Analysis:
Quantitative & qualitative analysis of HIV proteins/peptides or HIV Abs through the use of specific antibodies. - More accurate/sensitive than ELISA tests - More technical, labor intensive - Not adequate for screening large sample number
Discovery of HIV Neutralizing Antibodies
Rare HIV+ individuals are able to produce neutralizing antibodies naturally. - 1990s- 1st anti-HIV Ab discovered able to neutralize 40% of known HIV strains - 2009- Anti-HIV antibodies PG 6 & PG 9 neutralize 73% & 79% (respectively) of known HIV strains - 2010- 2 more naturally occurring anti-HIV Ab-named VRC01 & VRC02- isolated from blood of 60 year-old-black gay male (Donor 45) who was a "slow progressor" ~ In 2014, Donor 45 was HIV+ for 22 years & was still alive ~RARE B-cells producing both Ab were isolated from D45. - 2011- VCR anti-HIV Abs - similar to VRC01 & 02-characterized in 2 African Americans (Donors 74 & 0219) ~ All VRC Abs bind to the SAME (constant) HIV region: CD4-binding site in GP120 (This was the FIRST Achilles' tendon of HIV)
How are these South African HIV+ children non-progressors (to AIDS)?
Reduced immune activation Less infection of certain immune cells (long-lived central memory CD4 T-cells) which are able to replenish their stock within bodies (healthy due to constant supply of CD4 T-cells). Non-progressor children also have low levels of co-receptor proteins (CCR5).
NATs are VERY ______________ & are best suited for _________ diagnosis of mother to child transmission or follow up of viral load in HIV+ patients undergoing antiretroviral therapy.
SENSITIVE early
Serological Methods to Detect HIV
Several methods allow for the detection of HIV - indirectly (protein-based tests): detect molecules produced in reaction to HIV infection (antibodies/Ab) - directly (nucleic acid tests) from the patients' sera/serums: Direct tests detect HIV presence through its proteins (antigens/Ag) or genome/expressed transcripts (RNA) - Blood samples are required for all tests
HIV Home Testing Options
Since 1996, existence of Home Collection option for samples: Samples (blood) can be collected at home by the patient & sent out to an HIV testing facility (laboratory) Recently, rapid HIV tests from oral swabs or tiny blood samples can be performed by anyone at home & provide instant results (20-40 minutes) - Any positive results obtained from home tests should be investigated further in a laboratory setting OraQuick in-Home HIV Test (2012; by OraSure Technologies, Inc): Quick home HIV test available in most U.S. pharmacies. - Detects HIV-1/2 antibodies in saliva in 20 minutes (costs ~$13). - Another home version can also detect HIV-1/2 Ab in blood (40 minutes) BioSure HIV Self Test (2015; United Kingdom) - Home HIV test that is 99.7% accurate (~$45) - Uses a very small amount of blood (finger prick) to detect presence of anti-HIV Abs. - Results available in 15 minutes. - Approved for use in U.S. by FDA in 2016; More accurate than OraQuick
Epitope:
Specific part of the antigen (Ag) recognized by its cognate antibody (Ab- produced against said Ag) and allows for Ag-Ab binding.
Sterilizing vs. Non Sterilizing Vaccines
Sterilizing vaccine - A sterilizing vaccine would be ideal as it could completely prevent the organism from an infection by the agent - Research data show that this strategy, although common against many other viruses, CANNOT be used against HIV - Therefore all efforts are redirected to develop a vaccine which could prevent the symptoms of the disease from developing in HIV+ patients Non-sterilizing vaccine - Non-sterilizing vaccines DO NOT provide a complete protection from the virus (sterilizing immunity) but will decrease the viral load and slow or prevent the disease progression - HIV poses a big problem in vaccine development - HIV does not follow the typical evolutionary pathway - HIV evolved away from the most common immune response
Protein-Based Serological HIV Tests (2 types)
TWO types of protein-based serological HIV tests allow for the detection of antibodies produced against HIV (= anti-HIV Abs): 1) Enzyme-Linked Immunosorbent Assay (ELISA): quantitative & qualitative biochemical assay that detects antibodies directed against HIV antigen - Targets= proteins, peptides: viral Ag or anti-viral Ab (Ab & Ag are proteins). - Efficient technique for large scale screening (high number of samples 2) Western Blot Analysis: quantitative & qualitative analysis of HIV proteins/peptides or HIV Abs through the use of specific antibodies. - More accurate/sensitive than ELISA tests - More technical, labor intensive - Not adequate for screening large sample number
Problems Associated with ARV Drug Development
The ARV drugs (ARVD) should target the virus efficiently WITHOUT any cytotoxicity Identify biosynthesis (biochemical) weaknesses in HIV's life cycle - Multiple targets are best (drug cocktails) Specifically target the provirus (inserted HIV genome) in infected cells ARVD must be able to cross the blood-brain barriers ARVD should NOT damage the non-regenerative brain cells ARVD should allow for the treatment of opportunistic diseases apart from HIV infection (no drug interference) Relieve cumulative stress in the body (not make it worse with side effects)
Recombinant Protein Vaccine
The Ag is produced synthetically in recombinant bacteria Ag-encoding genes are inserted in a bacterial plasmid. The recombinant plasmid (viral + bacterial DNA) is introduced in bacteria for Ag production and purification. Purified Ag are injected as preventative vaccine A recombinant subunit vaccine was developed for the hepatitis B virus (Recombivax). A rec. subunit vaccine against hepatitis C virus is being investigated. - 2015: An efficient DNA vaccine Hepatitis C Vaccine (HCV) was developed in mice
Principle of the ELISA Test Capture competition test (capture assay sandwich)
The capture antibody (anti-HIV Immunoglobulin G, anti-HIV IgG) coats the inside of each well in a microtiter plate The capture antibody binds to the target protein (HIV antigen- a "piece of HIV"-; HIV Ag). The detection antibody (anti-human IgG- from another animal/horse etc...) is bound conjugated-through streptavidin/ biotin- to an assayable enzyme (HRP, HorseRadish Peroxidase) - A substrate processed by HRP creates a detectable & quantifiable light reaction showing the presence of HIV in the serum - Assay result is analyzed by chemiluminescence (light emission)
Position effect of HIV genome insertion (epigenetics)
The random insertion of HIV genome in T-cells' genome may also "silence" the provirus (no viral gene expression) - Heterochromatinization causes gene silencing- (chromatin condensation causes epigenetic regulation of gene expression.) - Proviruses inserted in heterochromatin regions are inactive -no viral gene expression hence NO HIV production- & also lead to creation of latent HIV reservoirs HIV infected T-cells harboring latent proviruses evade detection (ART treatment) Histone Deacetylases/HDAC inhibitors: HDACI - HDACI: FDA approved (2006) cancer drugs that block HDAC enzymes (promote de-condensation of heterochromatin i.e. compact chromatin.) 2012-2015 -In vivo treatment of HIV-infected human T-cells (cell cultures) with HDACIs induced epigenetic reactivation of provirus in latent HIV reservoirs (induced viral gene expression & virion production)
Viral Load and the Course of AIDS
There is a relationship between the HIV/viral load and the CD4 T-cell count in an HIV/AIDS patient. - Early in the disease, the viral load rises dramatically as the CD4 T-cell count drops (acute phase). - The viral load decreases as the immune system is engaged to fight the infection. - Eventually, the viral load rises again in the later stages of the disease as the patient progresses to AIDS.
Plant-Produced Vaccine Proteins
Transgenic plants are easily grown and are a source of recombinant proteins - Recombinant plant proteins from transgenic plants not yet approved by FDA Edible vaccines - Currently in clinical trials - Produced in food plants - Decrease production cost - Vaccine against cholera produced in genetically engineered potatoes
The U=U Campaign (since 2016)
U=U: Undetectable = Untransmittable (or Uninfectious) An undetectable virus load DOES NOT equal a ZERO virus load
Western Blot (WB) Analysis
WB can detect HIV antigen proteins or HIV antibodies (immunodetection with antibodies directed against HIV Ag or anti HIV Ab) - WB is more accurate though more labor intensive, costly & time-consuming - WB is a good confirmatory test for HIV positive samples, but NOT an efficient screening method. HIV proteins are isolated & separated electrophoretically by size in a denaturing (SDS detergent) polyacrylamide gel matrix (SDS-PAGE electrophoresis). Because HIV proteins are separated (gel=sieve) by size in WB, this technique is MORE specific than the ELISA. - Antibodies can interact with single HIV proteins or anti HIV Ab at a specific spot in the WB
Positive results of ELISA tests are confirmed with _________ ______ ____________
Western Blot analyses to reduce the instances of false positives (= HIV+ diagnoses when in fact HIV-) - Negative ELISA tests are NOT reconfirmed thus increasing the chances of false negatives (=HIV- diagnosis when in fact HIV+) - Chance of false negatives & false positives are extremely small. Because of the window period after infection (up to 6 month for anti HIV Ab production), a second HIV test is given 6 months after the first one. - Efficient to confirm HIV status if the person has not been re-exposed in the meantime.
There is now evidence-based confirmation that the risk of HIV transmission from a person living with HIV (PLHIV), who is on Antiretroviral Therapy (ART) and has achieved an undetectable viral load in their blood for at least 6 months is negligible to non-existent.
While HIV is not always transmitted even with a detectable viral load, when the partner with HIV has an undetectable viral load this both protects their own health and prevents new HIV infections.
While combination/combined drug cocktails are efficient initially, they do ...
also eventually breed HIV resistance and the rise of highly resistant HIV strains
The serum is used in many medical tests as it is a great source of ______________
biomarkers - biological molecules indicative of a specific medical condition - Presence of antibodies against a specific foreign agent indicates an infection status with the said agent.
Entry or fusion inhibitors
block the entry of HIV in the immune cells Co-receptor antagonists block co-receptors on target T-cell host Fusion inhibitors block the structural changes required for the viral envelope to fuse with the host (CD4 T-)cell plasma membrane - 2003: T-20 (Enfuvirtide, Fuzeon) $44,604/yr/person - 2007: Marivoc (Selzentry, 1st CCR5 co receptor Antagonist) $14,436/yr/person - Small number of CXCR4 (R4) and CCR5 (R5) blockers Problems Associated with ENTRY Inhibitors - Entry inhibitors like Marivoc (Selzentry) are only efficient against CD4+ cells using the CCR5 (or R5) co-receptor. - Cannot combat HIV efficiently since virus can infect CD4+ cells with CCR5 or CXCR4 (or R4) co-receptors. - R4-targeting HIV is more common in HIV+ who have been infected for a long time.
People living with HIV (PLHIV) in Kansas may be .....
charged with a felony for having sex with the intent to expose another to disease - It is a "severity level 7, person felony" for a person who knows they are "infected with a life threatening communicable disease" to have vaginal or anal sex with the intent to expose another person to the disease.
HIV Screening Methods
detect the presence of the HIV virus in the blood, either directly (presence of viral components: RNA or viral Ag proteins) or indirectly (presence of antibodies against HIV) - Sensitivity of each detection method differs - NO detection does NOT always rule out virus presence Two types of serological tests based on different detection techniques - screening test and confirmation test- are used to evaluate any HIV infection status When the results of a screening test are positive or indeterminate for HIV, a confirmation test must be successively conducted to confirm seroconversion. Any positive confirmation test ultimately validates an HIV infection status (person is HIV seropositive). - The HIV+ patient, if treated (ART) may NOT develop immunodeficiency for years after seroconversion
The serum
is the clear bloody fluid obtained from blood after coagulation i.e. after removal of any blood cells (white or red- WBC & RBC and clotting agents/fibrinogens).
Two types of serological tests based on different detection techniques - __________ _______ and _____________ _______ - are used to evaluate any HIV infection status
screening test and confirmation test
Recombinant plasmid=
plasmid with inserted foreign gene
Viremia
refers to the presence of virus in the blood
When the results of a screening test are positive or indeterminate for HIV, a confirmation test must be successively conducted to confirm _________________
seroconversion
Once anti-HIV antibodies are produced (& detected), the infected person has ________________
seroconverted = he/she now tests HIV+ (HIV seropositive).
All HIV tests are _________/__________-based
serum/blood
The choice of a specific test over another will depend on the ______ of the HIV infection
stage
difference between reverse transcription and Polymerase Chain Reaction
template is a single strand RNA and the polymerase enzyme is a reverse transcriptase (RT). Short DNA primers (FWD and REV like in PCR) are used in the RT-PCR reaction which is also completed in a thermocycler. HIV's RNA is directly detected using RT-PCR
The seroconversion window period is ......
the time between HIV infection & anti HIV antibody production by the infected individual. (person will test HIV- during this period)
