MIMM466 Final
NEF involvement in pathogenesis
1) Animal models showed that NEF depleted strains of SIV didn't develop disease in macaques. Those that developed disease had mutations in NEF gene that reverted. Also tested in vaccine related studies too with protection when later challenged with WT virus. 2) Natural infection Long term survivors or nonprogression was looked at for what mutations were important. CCR5 or sepcific HLA were helpful. In 8 people infected from a single donor they had NEF defect or LTRs with time more mutations made them more infectious others died from non related causes. after 26-29 years of infection 3 are LNTPs and the donor has moderate HIV assocaited dementia 3) Trasngenic mice: expressing the entire HIV1 genome in CD4 T cells and different forms of mutants. which induced an AIDs like disease. Only those with the intact NEF induced an AID's like disease. So NEF has a major determinant of pathogenicity in AIDs.
The seven priorities towards HIV cures
1) Molecular biology of HIV latency 2) Immunology of HIV persistence 3) Models for HIV cure or sustainable remission 4) REmission in pediatric populations 5) GEne and ell thearpies 6) Novel biomarkers for HIV persistence 7) Assocaition with social science and health care systems research
The transformation of AIDS to a chronic disease
10% have virological failure 20% with immune chronic inflammation Need to treat lifelong due to the remaining viral reservoirs Have to deal with side effects like premature aging, cardiovascular neurological disorders, cancers, liver and kidney failure: all co-morbities.
VISCONTI cohort
14 patients diagnosed with HIV in late 1990s to early 2000s and were treated within 10 weeks of infection Some stopped therapy after 3 years. Some had reduced weak viral reservoir reaching remission.
Vancouver HIV patient
18 year old woman had gone into HIV remission for 12 years. Born in france to an HIV poistive mother and was treated with AZT and diagnosed HIV+ at one month. Treating at 2 months with 4 antiretrovirals follow up to 6 years old. and At 18 years old she has small blips but virus generally not detectable so she'd gone into "remission."
History of AIDS from 1930 to 1989
1930 - passed SIV to humans starting the pandemic. 1981 - first few cases in gay men (gay plague) 1982 - Disease reported in Europe and America. Coined AIDS or SIDA 1983 - heterosexual contact also transmits it 1984 - Isolated by the retrovirus in France then demonstarted to cause AIDS by robert gallow. known as HTLV3 at the time Europe reports on 2 AIDS pandemics in homosexuals and central Africa immigrants (7,700 cases in US and 762 in EuropE) 1985- developed the blood test with seqeuncing done. Lots of fear around these things. First AIDS conference was held. and Reagen mentioned it in public for the first time 1986- Renamed to HIV 1987 - AZT introduced as the first antiretroviral as a reverse transcriptase inhibitor Some argued that HIV was not the cause of AIDS and princess diana shook hands with an AIDS patient. 1988 - First world AIDS day 1989 - second anti retroviral of DDI developed as a nucleoside ART inhibitor.
History of AIDS from 1990 - 1998
1990 - Ryan white dies Worldwide 8-10 million people infected 1991 - Red ribbon becomes symbol for AIDS DDC approved as the first time AZT and ddC were used as a combination therapy 1992 - India funds national AIDS controls with over 15% of its health budget 1993- some viruses are found to become resistant and mutations can transmit in the population 3Tc approved for clinical use Incredibly important for first line treatment 1994 - AZT was shown to reduce mother to child transmission of HIV 1995 - first HIV protease inhibitor was approved. 1996 - First use of HAART with great clinical response Identified CXCR4 and CCR5 as coreceptors for HIV. First non-nucleoside reverse transcriptase inhibitor was approved. 23 million infected worldwide 1997 - latency of HIV was described. Drop in deaths occur because of HAART. Approximate infected jumps to 30 milllion 1998 - Azt price cut to protect trasnmission from mother to child VAXGEN clinical trial for vaccine was started but eventually failed also found serious side effects from VaxGEn also found transmission of multi drug resistant HIV documented.
HIstory from 1999 to 2003 of AIDS
1999 Traced HIV to Chimp virus Nevirapine showed reduction of mother to child transmission of HIV. Huge increase because of needle sharing in Soviety union 2000 - first international AIDS conference in South Africa CIA talks about threat of AIDS Denial of AIDS in south Africa occurs but Botswana estiamtes one in four adults are HIV positive By end of 2000 34.3 million people were HIV positive. 2001- HIV budding shown to involve vsicular transport China acknowledged the threat of AIDS to its public health. Lots of efforts from the government to fight the HIV pandemic. 2002 - Described the non random integration of HIV. Ukraine reports 1% of population is HIV positive And HIV super infection was documented also found APOBEC3G found 2003 - The new class of antiretrovirals developed of fusion inhibitors. 14K people infected with HIV every day, 40% postiive population in Swaziland Presidents emergency plan for AIDS relief done. Vax gen finally fails
AIDs history from 2004 - 2006
2004 - TRIM5 alpha reported for preventing HIV infection Botswana provides treatment to half of the individuals. 2005 - Nelson Mandelas son dies of aids Death from AIDS decreases generally. Rate of infection goes up particularly in African Americans. Treatment was used as prevention for infected individuals here. And now transfer of HIV is lowered. Russia increases AIDs program spending 20 fold Generic cheaper AZT was approved 2006 - Male circumcision reported to decrease transmission by 50% from infected women to men Atripila pill was approved for sale with one formulated pill. Nearly 40 million people were infected 25 million people had died of AIDS
NEF protein structure
27 kDa protein that is phosphorylated. Produced from early to late phase of infection of HIV infection. Gets localized to the PM and cytoskeleton and they get incorporated into the Virion
Total Infected by AIDS
36 million
HIV vaccine trials
5 completed the 3 stages No efficacy and some with negative effects. 31% prevention at p= 0.04 for the Thai trial (RV144) But was successful because of no association with neutralizing antibodies, cellular immune responses. And Decreased risk associates with IgG antibody responses to the V1/V2 loop with ADCC activity in the C1 region of envelope And low IgA Antibody responses Then efficacy was in a low risk population and faded with time. but the effects weren't great.
Oral DTG for TasP
86% effective to prevent infection because resistance never reported in clinical therapies. With resistance against NRTI, and which it has been coutilized has never been reported with DTG. Mutations for DTG confer low level resistance with greatly diminised viral replication capacity. Likely incompatible with viral survival. and also there's no compensatory mutations. and has a long half life of mutations. Also one of the safest and best tolerated drugs in the armamentarium and can be coformulated.
Downmodulation of CD4 in HIV-1
A hallmark of HIV infection which is correlated with HIV-1 Pathogenicity. Not only mediated by NEF but also by others. Env binds to CD4 in the ER and cell sruface before translocation to seqeuster it. And also VPU and NEF affect the intracellular trafficking of CD4.
What is ADCC and HIV's resistance
ADCC is the antibody dependent celular cytotoxic response. So NK cells kill the infected cells. And in the surface of NJ cells that have FC receptors. When they bind to the FC portion of the antibody and they bind the envelope on the surface of the viral particle. Other recpetors and ligands are engaged and then the NK cells are activated secreting lots of enzymes to kill the infected cells. Has the potential to reduce the HIV reservoir. HIV can respond with sequestration of CD4 and also secrete gp120 which will bind to uninfected CD4. So now NK cells will kill CD4 T cells which are not infected. BUt if you can open the envelope it'll make it more susceptible and enhance the ADCC effect.
TAT, DEmentia and Central nervous system
AIDS associated with dementia in about 20% of patients mostly in late stage of the patients with monocyte and macrophage infiltration in the brain. TAT acts as a cytokine and chemoattractant for monocytes. They induce production of neurotoxic, and excitation cytokines and general neurotoxicity. So HIV infected macrophages can infiltrate the brain and TAT exits the cell it produces and release envelope. Infected macrophages and microglia release those cytokines like TGF-beta, IL-1 and TNF alpha. Tat also can activate uninfected macrophages and microglia and produce some cytokines. Also the major cell population of 80% astrocytes come in contact with these and produce TGFbeta and can also become infected. They produce a low level of virus and affect the neuronal damage and apoptosis which lead to HIV induce dementia.
Improvement of antiretrovirals over time
ART (Antiretroviral triple therapy). Fast development over time. Low potency high toxicity originally. Eventually improved upon these drugs potency and but still high toxicity. Now high potency and low toxicity. Normalized survival increasing age of survival to normal of normal people. (but it doesn't eliminate) (Antiproteases were the beginning of triple therapies and afterwards viral load started dropping rapidly in 2008)
Recommended Anti retroviral regimen
ARV usually consists of two nucleoside reverse transcriptase inhibitors (NRTIs) in combination with a third active ARV drug from either integrase strand transfer inhibitors (INSTI), a non-nucleoside reverse trasncriptase inhibitor (NNRTI) or a protease inhibitor (PI) with a pharmcokinetic (PK) enhancer booster (cobicistat or ritonavir)
SINE structure
Also have other short retrotransposons. Range of 500-1000 bp and noe encoded protiens They need the help of the proteins from LINe To complete reverse transcription.
Cabotegravir
An analogy of dolutegravir which has a long half life. Suppresses the virus to the maximum level and stop the treatment. With Intramuscular cabutegravir. And another drug in the vein Can be taken bimonthly or monthly. So patients can suppress the virus and with rilpivirine and lamivudine. So this is a potential for long lasting drug that can last a long time.
Curing ART with dolutegravir
An integrase inhibitor and resistant strains are not a very fit form of HIV.
Ethics concerns in Preventative HIV vaccine research
Because we have effective PrEPs, is it ethical to recruit volunteers considering the success of PrEP when vaccine candidates may not work
The gene therapy strategy for functional cure of HIV
Berlin patient was cured with CCR5 delta 32 mutation with transplanted HSCs. but 20% die from transplants and these mutations are rare. So the idea is to transduce the cells for an anti HIV or short hairpin RNA and have CCR5 delta 32 mutation and then reinfuse them into the patient. so either use an HDV ribozyme or shRNA for this for antiHIV functionality.
VPU prevention of cell surface expression of CD4
Binds the cytoplasmic tail of CD4 and binds another complex of AP-1. Thie medaites teh degradation through the ubiquitin proteasome pathway.
NEF prevention of cell surface expression of CD4
Binds to AP2 and mediates the internalization and brings the CD4 to endosomes and lysosomes for degradation from clathrin coated pits.
Env protein sequestration of CD4
Binds to CD4 during transport from ER to cell mbmrane. This prevents migration to the cell surface
Mississippi baby
Born to an HIV infected mother and medication was started within 30 hours of birth. But baby and mother stopped treatment and later the mother came back and the HIV didn't develop in the baby as a functional cure in 2.5 years old. But at 3.5 years the viral rebound was not cured.
Different binding sites of HIV-1 broadly neutralizing antibodies
CD4 binding site V1 V2 loop (stabilizes the dimer) V3 loop (corecptor binding point) gp120, gp14 interface and MPER (anchor region near the Transmembrane region)
Why was it wrong of the scientist to do the CRISPR editing experiment
CRISPR has unknown side effects Also not feasible to completely sequence the entire genome from a single cell before reimplantation of the cell Its in germ line And only should be applied to noncurable genetic diseases with zero alternatives also it wont for sure protect against HIV with CXCR4 strains and there are alternatives for protection And should only be used to treat lymphocytes because they wont be aboslute protection
Comorbidities or other conditions to HIV
Cardiovascular disease, liver disease, psychiatric illness, neurological disease etc. Pregnant and drugs for treating pregnant women Also if they have coinfections like HBV HCV and Tb.
Additional problems for HIV
Continued Viral Transmission Drug resistance Transmission of Drug Resistance Drug toxicities And Long term consequences of both HIV and antiretrovirals. (HIV associated neurological disorders) Especially suboptimal therapeutic regimens in developing countries leads to exacerbation of problems of selected and transmitted drug resistance in those countries.
Concerns for Developing and Developed countries for HIV.
Developed countries many fail most available therapies and for those who may have run out of treatment options with many resistant to drugs. Patients in developing countries don't have access to high quality reagents and mostly only have raltegravir in third line.
The special case for those who are HLA-B*5701 negative
Dolutegravir with Abacavir and lamivudine. Because in patients that do have that, they can get hyper sensitivity.
Functions of NEF
Down regulation of CD4, MHC1, IL-2, and CD28. UPregulation of Fas Ligand Also binding to multiple members of the src family kinases And NEF associated with kinases ASK1 and PAK
Why use Bnabs?
Drugs can block the infection or prevent spread of infection. But Bnabs work differently where they might bind to the envelope and neutralize and kill the viruses. Antibody bound virus particles have complexes that can engage the FC receptors on the macrophages or DCs with the APCs. Leading to engulfing of antibodies and the virus particle complex, presening small peptides and activating CD8 t cells. THis arm is not found for antiretroviral drugs. So this is used to treat chronic infected humans.
MK-8591 Novel Nucleoside
EFdA: A novel nucleoside inhibitor and has a unique method of action. Drug has a low EC50 of 0.2 nM and is a potent antiviral. non-obligate chain terminator, inhibits reverse trasncriptase preventing translocation. Was given to resistant monkeys and suppresses viral load quickly but it does come back over time. So this may have a long term potency with longer half life..
Prevention of HIV infections
Either for preexposure prophylaxis. A treatment of prevention (TasP) And vaccines. So for people that engage in high risk behavior they can take antiretrovirals to prevent infection At top efficacies you have 86% to prevent infections.
Humanized Mouse bNAb treatment studies
Either use one BNAb or 2-3 in combination for treating mice Can lead to 10-20 fold drops of viral loads. You get rapid emergence of the virus though because the virus can change the envelope quickly Especially with monotherapy with one BNab.
The major classes of antiretroviral drugs
Entry, Reverse transcription, integrase, transcription, virus assebly and production ,and protease processing. No transcirption inhibitors are approved for clinical use nor for assembly. Protease inhibition works by preventing maturation of HIV particles so that they're non infectious.
Importance of other prevention methods besides drugs
Even though PrEP has not promoted higer rates of STDs, you should still use caution and use condoms.
Genome make up of transposable elements
Haploid genome is 3*10^9 base pairs of DNA. 3-4*10^6 individual integrations of transponable elements are within that So for every 1 kb of DNA in the genome you had one event of transposable elements. In various animals you have different levels of each type of transposon or different amounts of each. (LTR, LINE, SINE, DNA , Helitron) Eg mice have a large percentage occupried by transposons compared to ecoli and plants have lower percentage too more LTRs.
HIV broadly neutralizing antibodies (concept)
Have various antigens on the surface of a viral particle that induces antibody production against them. So many B cell antibodies can be produced some are neutralizing and some are not. Some are specific to one or two types of envelope others broad. Generally neutralize many strains as broadly neutralizing antibodies binding conserved sites in the envelope. In patients this development is a long process taking 2-3 years normally. So now they've isolated quite a number of these different broadly neutralizing antibodies
Forms of transmission for ERVs
Horizontal transmission between one individual to another. from retroviral transmission and infection Eg HIV. Vertical transmission is where retroviruses will become an endogenous retrovirus in germline infection. This gets passed along generations and many ERVs are in the genome from long ago.
Models of bNAb studies
Humanized mice: Reconstituted with human immune cells in immunologically incompetent mice. - Then also use HIV-1 Macaques - more similar to humans but requires a modification of SHIV instead of HIV. Macaques are expensive and humanized mice are cheap but not easy to generate.
TAT and angiogensis
In Kaposi's sarcoma you often have have lots of highly angiogenic lesions with coinfection of HIV. TAT is a growth factor for KS cells and endothelial cells inducing the migration of both and angiogenisis in vivo. So TAT could be a candidate of the cause of KS in AIDS but eventually found HHV8/KSHV in every KS lesion so Tat is just a contributing factor. Eventually found that Tat is a tumor profressor and angiogenic factor contributing to the aggressive nature of AIDS-KS. VEGF tyrosine kinase receptor 2 is signaled to induce a tyrosine kinase pathway of MAPK leading to cell growth, migration and angiogenesis.
Tissue culture of viruses treated with Dolutegravir
Isolated a mutation of R263K and H51Y which after selection by DTF are impacted in fitness so they aren't observed in patients. Other mutations have good replication fitness but R263K dropping around 50% Making an evolutionary dead end.
REV RNA binding
It has two RNA binding domains/NLS+RRE bidning domains NLS: Nuclear localization signal NES: Nuclear export signal also there with activation domain. Rev starts in cytoplasm when synthesized. It'll get localized in the nucleus going through the nuclear pore complex Then the RRE RNA is synthesized in the nucleus and gets bound by REV. Its NES also binds the CRM-1 also known as exportin 1 or RAN GTP that help it bind to the nucleoporin in the nuclear port. Then with consumtion of GTP to GDP it'll go back to the cytoplasm. RAN GDP and CRM are then recycled. These can then get packaged into the virion too with the full length of RNA RRE.
Retroviruses in koalas
Koala retrovirus is associated with leukemia and lymphosarcoma in koalas. Northern part koalas carry more of the retrovirus and in the south the prevalence is very low. 150 vs 1.5 Looking at the koala retrovirus DNA you can see koala retrovirus in parents. T Use a southern blot to probe for koala retrovirus DNA. Looking at koala tissue see these patterns are identical. See these bright spots.of signals as they're passed down the generations and change. see them in germ cells through FiSH as well.
Retrotransposon classes, naming and genomic distribution
LTRs, LINE, and SINE LTRS - 9% (LTR Retrotransposon) LINE (Long interspersed nuclear elements)- 21% SINE (Short interspersed nuclear elements) 13% These are further subdivided. Lots more copies of retrotrasnposons in our genome than genes we have.
ERVs in evolution
Look for two species like humans and new world monkeys look for sahread ancestor and what ERVs are carried. So you can look back to determine the age of a retrovirus by seeing common retroviruses between other species.
Summary of NEF pathogenesis
Major determinant in SIV infected macaques, in HIV infected LTNPs, and in trasngenic mice Also leads to downregulation of surface molecules like CD4, MHC-1, IL2-R, CD28 and upregulation of Fas-L Nef also induces alterations in src kinases and ASK1 and PAK.
Vaccine development for HIV
Many years of research contributed towards understanding anti HIV immune responses, such as understanding for the development of a succesful vaccine to protect against HIV infection. But vaccine research must be placed in the context of other prevention research. So must consider drugs and other measures.
Examples of mammalian ERVs
Mouse, koala human etc. Have gamma retroviruses (Class 1) Beta (and one alpha retroviruses (Class 2) Spuma retroviruses (Class 3) Most human retroviruses are Class 1 Gamma. There's one beta human and 2 in spuma The ERVs that infect mammals are different distributed depending on the species. There are also MaLR (mammalian apparent LTR retrotransposons). Different patterns of the genome.
LTR transposon structures
Much like the retrovirus, LTR is on each end. and some still encode Gag pol and envelope But many have also mutated and deleted those viral genes.
Measuring the HIV-1 reservoir.
Multiple intermdeiates like DNA, RNA, proteins or virions to measure. Each could show a different sized reservoir. Also only 11% of cells have an intact genome.
How does NEF lead to its functionalities
NEF interacts with the cytoplasmic tails of CD4 of MHC-1 and AP2 which is in clathrin coated pits These form into endosomes and then are often degraded with lysosomes.
What RNA targets are there against HIV?
Only target in clinical trials is at 5983. Want to select areas also in highly conserved regions too. Also one in the gag region too. They showed the ribozyme decreased viral replication at that gag region at 1498 with shRNA also decreasing it further But it was insufficient to completely inactivate. But it does provide a more complete inhibition of viral production. But regardless found 1498 shRNA was more active than the other shRNAs against other regions.
Regulation of HIV Translation.
PKR is only transiently activated after HIV infection and is deactivated during high viral replication HIV replicates in cells that produce large amounts of TRBP which release the block of translation caused by TAR and counteract the PKR activation. HIV infection will also increase the production of ADAR1 which inhibits PKR. PACT will become a PKR inhibitor in those HIV infected cells. All leading to the increased translation of HIV in cells.
What patient factors are important to persons with HIV?
Pretreatment HIV RNA level/viral load. CD4 count. HIV drug resistance testing results (so is it a specific genotype?) HLAB5701 status Individual preference. Anticipated adherence to the regimen
Gatignols opinion of generating effective host immunity for functional cures
Proof of principle from the Berlin patient So need to look at CXCR4 or virus. Replacing the HSCs in gene therapy might be enough. Also how you express permanent antiviral molecules (but can't otherwise you migth become immunogenic) Also looking at memory T cells for funcitonal cure. Also can avoid the brain you don't get infiltration cause astrocytes don't produce virus So just kill main reservoirs in blood. Also have new strategies like Zinc finger endonucleases, CRISPR. But they have delivery problems because you can't permanently express them.
HIV-1 Nucleosome Organization
Provirus is integrated into human DNA and organized into nucleosomes Important initiating factors including SP1, NFKappaB and TBP (which binds the TATA box) When in closed conformation it can't be transcribed so the regulatory factors help bind to initiate formation of TAT.
What are endogenous retroviruses?
Repetitive DNA in the human genome. Often seen in pieces. The nuclear genome in the nucleus at the DNA sequences. Quite a few are repetitive with thousands of DNA repeats. They can play centromeric repeats and make centromere in our chromosomes. Also telomeres with telomeric repeats. Also have dispersed repeats along the genome Two classes (one and two) Class two transponsable elements are DNA transposons. Class one being retrotransposons. Going from RNA to DNA in cycles. Among these is retroviruses and LINEs.
The different forms of cure or remission of HIV-1
Sterilizing cures - The elimination of all latently infected cells by shocking and killing (usually work through reactivation of latently infected cells.) Functional cures where you maintain infected cells in a permanent latency Remission is non detectable virus after treatment interruption.
The recent advances for different strategies for treatment of HIV
Sterilizing shock and cure Gene therapy functional cure. Anti TAT molecule for functional cure or deep latency. Dolutegravir for functional cure or deep latency And Early treatment for long term treatment.
Mechanism of HIV-1 of REV
Synthesized from the doubley spliced exome of exon one and two. In REV deficient viruses you could see RNA of doubley spliced RNA at only 2 kb. (no 4 or 9 kb) So low Rev gives you synthesis of the long RNA which is then spliced down to 2 kb. but in late phase with Rev you have the structural RRE thats on the long RNA which when bound cannot be exported and same with singley spliced. So TAT increases your tranacription and REV in late phase helps negate export of RNAs. long 9 kb will help encode Gag and Gag-Pol While 4kb will encode Env, VIF, VPR, and VPU.
Translation of HIV-1 RNA
TAR structure inhibits translation of HIV RNA. RNA will go to the cytoplasm where TAR is no longer present. A protein kinase in the cell that is activated by dsRNA when activated will become phosphorylated with translation initiation factor alpha. You have Tar bidning protein (TRBP) that bind TAR and inhibit PKR. Releases the structure of the block. PACT normally activates PKR is also blocked by TRBP And then ADAR1 also inhibits PKR. So TRBP releases that block of translation due to TAR.
HIV-1 Expression through TAT
TAT gets translated from spliced RNA and then is translated from the binding of the two sequences in the cell. Doubley spliced RNA is used for the exon and second. TAT acts on RNA binding to TAR RNA in the structure. So this TAR will be set aside on the 5' end of the RNA and mediate TAT transactivation and play roles in translation.
Expression of provirus
TAT is translated from spliced RNA by binding two sequences in the cell with doubley spliced RNA.
TAT and TAR
Tat acts through binding to an RNA element that is a trans activation response to RNA. TAR has a stem bulge loops that has several domains like acidic, cysteine, core and RNA binding domains. It reaches the arginine here which is important for transactivation activity. With basal transcription you have TAT box with BDAF but with pol II all factors join. initiation phosphorylation on the polymerase II's CTD. TAT recruits pTEFB to the promoter to enhance trasncriptional elongation through hyper phosphorylation.
Extracellular influence of TAT
Tat has many functions including exit from the cells that produce it from transfected cells, entering into cells in culture or activate HIV LTR in neighboring cells. It'll also interact with many receptors on cell surface
Summary of TAT for HIV pathogenicity
Tat is required for viral replication due to its transactivating activity Angiogenesis induction in endothelial cells andAIDs-KS. Also in AIDs associated dementia through direct toxicity, cytokine production and attracting cells like monocytes and macrophages
Barriers to curing HIV-1
The HIV integrates into the human chromosome as a provirus. Need to find ways to eliminate a provirus or permanently inactivate it or to eliminate cells that have it. And it'll hide in anatomical reservoirs like atrocytes and microglia, lymph node lymphocytes, gut and genital tracts. With both active and latent reservoirs with residual replication or in T cells with survival or proliferation. Its hard to distinguish latently infected cells from uninfected cells. They persist in local tissues. And highly diverse populations.
Basal Transcription of HIV-1
The chromatin is opened by binding SP1, NFKappaB and TBP (to TATA) this gives nucleosome remodeling. Mainly mediated by NFKappaB which allows TAT to be made. Tat has a feedback loop that helps to open the nucleosome.
Two approaches to HIV cures
The elimination of all HIV infected cells - Sterilizing cures Or the Generation of effective host immunity to HIV that would result in lifelong control of the virus without therapy (functional cure)
HIV treatment regimen specifics
The genetic barrier to resistance Potential adverse effects Potential drug interactions with other medications Convenience (pill burden, dosing frequency, availability, or fixed-dose combination formulas and good requirements. Cost (some are too expensive)
The "Berlin" patient
The only person cured of HIV. Had HIV for 20 years and got acute myeloid leukemia after responding well to ART. He needed allogenic bone marrow transplant and got pretransplant conditioning that they got irradiated. Transplanted with an HLA matched donor who was homozygous for a CCR5 defect delta 32 which is found in some northern europe populations after irridiation.
Other targets for gene therapy
The transdominant RevM10 Antisense envelope entry inhibitors like Peptide C46 And combinations of restriction factors. You also have other strategies using endonucleases or CRISPR to target CCR5 DNA or viral genes.
Cornerstones of HIV prevention research
The understanding of social, societal and political factosr that contribute to HIV spread The distribution of condoms and decriminalization of HIV infection 2nd is treatment as prevention and the WHO hsa a 90 90 90 gola There is financial incentivization of HIV testing. 3rd is Pre-exposure prophylaxis (PrEP) (Truvada once daily had 86% effectiveness) on demand Cabotegravir or Dolutegravir Or EFdA. 4th) Microbicides with some stories that are useful. 5th) HIV vaccine research with more failures than successes. Looking at broadly neutralizing antibodies (exposure of epitopes and modification of envelopes and conserved envelope epitopes) About immune function like T cell killing. And many approaches involving the use of conserved envelope epitopes as targets.
Using CRISPR in HIV cure
There is viral escape in single gRNA therapy but got cure from dual gRNA CRISPR therapy. So want to deliver CRISPR but need a mechanism to prevent it from being immunogenic cause it'll be permanently on. So might deliver through adeno assocaited viruses etc. And also want to reach all the reservoirs. Might not be enough to just block T cells. But it still decreases viral burden on monkeys by targetting CCR5 and the PD-1 blockade So it might be a better strategy to kill the reactivated cells with PD-1 targetting.
HERVs
These are inactive and don't transpose in human genome unless in certain conditions. Some of them are transcriptionally active during embryonic development and in pluripotent stem cells like cancers.
The human endovirome
These are the viral seqeunces in our genome Have around 650K ERVs in the human genome 577K called solo LTRs 70K truncated proviruses. have lost part of their viral gene between the LTRs 10K with two LTRs flanking internal sequences. 2000 of the HERVs are more than 3.5 kb. only one HERV (HML-2) shows recent activity. The rest of all retroviruses in our genome are inactive. Many have lost genes and the retroviruses. Are passed along the the human genome.
Primary mutations of HIV for INSTI Resistance
They confer resistance to the drug and can reduce viral infection. The viruses can come up with compensary mutations to recover fitness in other mechanisms But compensatory mutations with DTG resistance and viral fitness have developed over 5 years in culture.
LINe structures
They have the LTRs and have two open reading frames. Encodes proteins that help to jump from one side to another ORF 2 will encode the reverse transcriptase and helps reverse transcribe the LINE RNA into DNA. LINE does not have the integrase which retroviruses have.
How do you select a drug regimen for a patient based on the number of options available of antiretrovirals?
Things to consider: Virological efficacy, toxicity, pill burden, dosing frequency, drug drug interaction potential, resistance testing, comorbidiy, access and cost. Eg: one that is one pill per month or one pill per week better than one pill a day.
Macaque BNab + ART treatment
Treating acutely infected Macaques. Without BNAbs the viral load goes up and is quickly controlled. So they might have elite controlled phenotypes where their immune system can contorl the virus at low levels so that they don't need further treatment to survive. VS in ART you get remission.
Is the Boston patient reproducible?
Tried to reproduce it and one survived for one year. but tehy were transplated with HLA donors that were WT for CCR5. Also other HIV can use CXCR4. SM patient had same process but had CXCR4 HIV which progressed to hodgkin's lymphoma and he died from the lymphoma.
Models for amplification of ERVs
Two models: one like retroviruses where the ERVs express their genes Make virus particles then same cells or another cells and then reverse transcribe. get back into the DNA also amplified. Second model: thought to occur because of mutants that have altered gag targeting or loss of envelope They undergo reverse transcription in the same cell So it'll insert new copies of its genome into the DNA and also amplify them.
Macaque BnAb studies
Used antibodies that worked well in humanized mice in the macaques Checking for emergence of resistant viruses Found great effectiveness of BNabs with how viruses respond and if they get resistance. With lengthy suppression and some having no rebound.
Clinical bNAb Treatments
VRC01 is one of the main ones used which binds to hte CD4 binding site in addition to two other BNABs All used as monotherapies in the tests. All cases saw brief reductions in viral loads but quickly led to viral escape.
Anti TAT molecule
Viral tat is a target as well Have a cortistatin A that is used in cancer from a sponge And it binds teh basic domain of TAT and inhibits TAT Tar interactions which induces deep latency. so basically you cannot activate the virus potentially Used both dCA and ART but found there were rebounds at Day 16 rather than day 7.
The HIV-1 Replication Cycle
Virus enters through CD4. (Coreceptors of CXCR4 or CCR5) Then you have absorption and entry and form the genomic RNA. Have reverse transcription making cDna. It gets localized in the nuclei and integrated. cDNA is integrated and then has transcription splicing with different splicing events. This can go back to the nucleus or is translated to make the protein together with the genomic RNA You then get virion assembly at the PM to bud and form mature particles to form a new virus.
Shock and Kill concept
Virus shown to persist in things like monocytes and macrophages too where they're unexpressed. Also in microglial cells macrophages and astrocytes in the brain. So they want to activate the virus from latency and then activate the immune response to kill the cell or increase ART to kill the virus. But they don't know how many are reactivated from killing T cells.
Difference betweeen HIV cure and remission
When the viral load drops below detection levels. When diagnosing them you put them on treatment quickly usually have rebound in less than 2 months but timothy brown had no rebound so he was cured. Vs the other patients that had rebounds at 3 and 8 months. And the mississippi baby for 28 months. That was temproary remoission While VISCONTI and french young woman were cured.
HIV-1 Genome Structure
You have the provirus, regulatory proteins and accessory proteins. Regulatory: TAT: Transactivtor of the HIV promoter REV: Nuclear export of late unspliced RNA to the cytoplasm Important accessory proteins: VPR: Induces G2 arrest and nuclear import of preintegration complex NEF: downregulate the surface CD4 and MHC1 (giving more infectivity) VIF: Virion Infectivity factor. VPU: Enhancement of virion release and CD4 degradation by targeting it to the proteasome
How did they understand PKR's influence?
You see peaks of virus when measuring trasncriptase activity Around Day 10 which you get reverse transcription yo monitor the phosphorylation of PKR. And PKR drops With TAR activation you get poorer translation. Then you can't deactivate translation anymore. Also get increases in ADAR. p150 wont get into the cells.
Finding the TAT complex with TAR RNA
pTEFB which is made of CDK9 and CycT. In a RNA gel mobility shift assay. The TAR RNA is labelled and the when you add TAT you get a shift. And with Cyclin T and CDK9 you get a super shift so that shows they combine together. TAT activity results in new transcriptional activation and gives processive elongation instead of the non proccesive of just just RNA pol II through hyperphosphorylation. Then absence of NF-KappaB or pTEFb (Cyclin T and CDK9) contributes to HIV latency in reservoir cells.
Therapeutic vaccine research for HIV
so should this be the focus You have two major pursued areas for HIV functional cures of Broadly neutralizing antibodies and T cell immunity. to improve and eliminate the infected cells. Leading to cures and functional cures instead.
The importance of dolutegravir clinically
the ATR single pill was less effective than DTG with the ABC and 3TC inhibitors. To some extent is superior to atripla also Dolutegravir has no major resistance mutatoins and no minor resistance mutations in treatment naive patients unlike raltegravir and elvitegrivir which have multiple resistancem utations So it has high genetic barriers for the virus to generate resistance to dolutegravir.
Options for a sterilizing cure according to Gatignol
use latency reversing agents like HDAC inhibitors or PKC agonists to release all virus and kill cells through intense ART. - sadly only partial activation is usually accomplished thorugh these methods not eliminating them so they need CTL activation in addition. - its also impossible to kill all infected brain cells - its difficult to predict and reach all reservoirs And killing all those reservoirs may affect another function. So maybe might be possible with CTL activation. Also some hope with CD32 to find latent cells.
before and after ART in HIV
without treatment you have gradual decline of CD4 in blood with rise in HIV RNA (acute phase) Then in the chronic asymptomatic phase you have plateau of HIV RNA but continued decline of CD4 when CD4 levels continue to drop viral RNA becomes more or less stable Then when below 200 CD4s the Virla RNA shoots back up and AIDS begins AFter ART, the viral RNA immediately drops and CD4 in blood gradually recover so that hey can live a normal ish life.
