In ancient times, citizens sought answers to their most important inquiries by consulting oracles. Myths were part of daily life, and mythological creatures People living with HIV/AIDS were feared or defeated. Oedipus deciphered the riddle of the Sphinx, a 34 million creature that was part primate (human) and part feline (lion). In modern times, people living with HIV/AIDS worldwide in 2011 many astute scientists have worked diligently to decipher a modern riddle, Number of people (all ages) living with HIV i.e., understanding human immunodeficiency virus (HIV) and using this Mortality knowledge to halt the AIDS pandemic. Although we endeavor to solve this 1.7 million riddle with the tools of science, we should keep in mind that, like Oedipus, we people died of AIDS-related illnesses worldwide in 2011 may find clues hidden in the intricate biology of primate and feline lentiviruses. Number of deaths due to HIV
Going Wild: Lessons from Naturally Occurring T-Lymphotropic Lentiviruses. VandeWoude & Apretrei Clinical Microbiology Reviews 27:728 (2014) Retroviruses • Over 40 nonhuman primate (NHP) species harbor species-specific simian Subfamily: Spumaretrovirinae (1 Genus) immunodeficiency viruses (SIVs). Subfamily: Orthoretrovirinae (6 Genera) Genus: Alpharetrovirus (9 Species) • More than 20 species of nondomes c Genus: Betaretrovirus (5 Species) felids and African canids demonstrate Genus: Deltaretrovirus (4 Species) seroreac vity against feline Genus: Epsilonretrovirus (3 Species) immunodeficiency virus (FIV) an gens. Genus: Gammaretrovirus (17 Species) • Rarely detected increased morbidity or Genus: Lentivirus (9 Species) impaired fecundity/survival of naturally infected SIV- or FIV-seroposi ve versus -seronega ve animals Len viruses • Cross-species transmissions of these Species: Bovine immunodeficiency virus agents are rare in nature Species: Caprine arthritis encephalitis virus • len viral infec ons have been Species: Equine infectious anemia virus iden fied in horses, goats, sheep, and Species: Feline immunodeficiency virus ca le, the majority of these infec ons Species: Human immunodeficiency virus 1 appear to be clinically silent Species: Human immunodeficiency virus 2 Species: Puma lentivirus • Sheep and Goat len viral infec ons Species: Simian immunodeficiency virus may result in neurological disorders, Species: Visna/maedi virus arthri s, and pneumonia, while horse len viral infec ons result in recurrent fever and blood diseases HIV-1 Entry • HIV, and other primate lentiviruses, infects CD4+ T lymphocytes and will cause them to die during a productive infection. • HIV infects other cells but does not cause cell death, at least at the same rate as CD4 T cells: – Natural killer cells – CD8+ killer T-cells – Macrophages – Cell of the nervous system (e.g., astrocytes, neurons, glial cells, and brain macrophages) – Dendritic cells HIV Entry
• The infectious virus is always specific for CD4 and CCR5 present on memory T cells and macrophages • The rapid progression to AIDS can be associated with a switch in co-receptor preference to CXCR4 • HIV enters cells via a cellular receptor and co-receptor – Major cellular receptor: CD4 present on T-lymphocytes (also present in low concentrations on macrophages) – Co-receptor on T lymphocytes: CCR5 (memory) or CXCR4 (naïve) – Co-receptor on macrophages: CCR5 Schematic of HIV Virion
Different HIV particles have highly variable protein spikes, Glycoprotein 120 or gp120 and random mutations in the HIV genome causes random changes in the structure of the spikes on different HIV virion. Extends out from the viral envelope as a trimer. The CD4 molecule interacts with gp120 The structure of CD4 CD4’s role in antigen recognition The union between HIV and its host T-lymphocyte GP120 trimers bind with CD4 and change shape gp120 interacts with CCR5 or CXCR4 (fusins), causing further shape changes and causes the viral gp41 trimers to be exposed
The gp41 molecule darts out and pierces the cell membrane of the macrophage or T-lymphocyte.
CD4 Viral envelope and cell membrane lipids fuse, delivering the HIV nucleocapsid into the cytoplasm HIV membrane fusion
The infec on with HIV1 isini ated by the interac on of viral gp120 envelope protein with CD4 protein expressed mainly by cells of the T lymphocyte and macrophage lineages. The interac on between gp120 and CD4 induces a conforma onal change in gp41, resul ng in the inser on of the N-terminal hydrophobic fusion-pep de region into cell membrane. Further intra-protein interac on between the N- and C-terminal of the gp41 ectodomain regulates the membrane fusion and the entry of HIV contents into cytoplasm. Besides the CD4 receptor, coreceptors CCR5 or CXXR4 also help the entry of HIV. The binding of gp120 to CD4 and its affinity for coreceptor largely depend on the sequence pa ern of its V3 and V4 variable regions. The replication cycle of HIV 1 and 2
Uncoating Removal of the genome from the nucleocapsid. Cellular enzymes strip the protein capsid away and the genome is released.
Capsid removal When the capsid is removed, RNA, two proteins (p24 and p17), and reverse transcriptase are released
Synthesis of viral nucleic acid and protein ssRNA is reverse transcribed into a dsDNA strand, which migrates to the nucleus of the host cell The DNA molecule is incorporated into the cell’s DNA at a random site by integrase, and becomes part of the cell’s 46 chromosomes. The viral DNA molecule within the chromosome is termed a provirus. This phenomenon is known as lysogeny The replication cycle of HIV 1 and 2 Assembly The provirus DNA is transcribed into RNA. Some is mRNA that is translated into viral proteins (e.g., gp120, reverse transcriptase, etc.). Other full-length RNA strands are used to construct a new generation of HIV genomes. Genomes, capsid proteins, and enzyme molecules are assembled at the edge of the cell, forming a circular structure that binds to the cell membrane
Protease snips out enzyme molecules and capsid proteins, then sections the capsid proteins into segments that unite and form an icosahedral capsid. Collapses to yield a conical capsid surrounding the RNA genome and enzymes. Note that p120 and gp41 extend from the cell’s membrane as spikes. The assembled viral nucleocapsid localizes to just under the membrane of the cells and buds through the membrane to form the envelope The replication cycle of HIV 1 and 2
Budding Virus coats itself with the cell membrane and pinches off, taking the membrane and spike proteins with it as the envelope. The virus is then released to the extracellular environment and HIV replication is complete. The capsid and matrix proteins and HIV enzymes need to be cleaved by a protease inside of HIV for the virus to be infectious.
Adapted from Vella, S., et al., AIDS Soc. 4 (1996): 15-18. Electron Micrograph Showing Mature HIV Particles
Courtesy of Louisa Howard, Dartmouth College, Electron Microscope Facility Figure 9-13 Basic retroviral genome HIV genome Know what gag, pol, and env encode, pls
Important drug targets
burst of replication when infected T cells are stimulated
necessary for the reverse transcription of RNA to DNA
enhances the movement of genetic messages into the cytoplasm One of the normal functions of the nef gene is to downregulate host antigen presentation. It does so by binding to HLA class I receptors via its binding motif thus interfering with intracellular HLA class I antigen processing Figure 9-15 part 1 of 4 Figure 9-15 part 2 of 4 Figure 9-15 part 3 of 4 Figure 9-15 part 4 of 4 Primary (acute) HIV infection Fever, pharyngitis, adenopathy, and rash Measures of virus and symptoms decline precipitously with seroconversion Early infection, virus completely homogeneous, late infection, viral heterogeneity
Immune response to Loss of CD4 T virus or loss of cells and CD4 memory opportunistic cells infections CD4 counts with time for individual patients (months after onset of primary illness) 32 years!
B T Grenfell et al. Science 2004;303:327-332 Viral infec on results in produc on of INTERFERONS
re noic acid inducible gene-1 (RIG-1) dsRNA-dependent protein kinase (PKR)
The host type I interferon response to viral and bacterial infec ons Andrea K PERRY, Gang CHEN, Dahai ZHENG, Hong TANG and Genhong CHENG, Cell Research Restriction factors = proteins that prevent viral replication, transmission, etc.
1. Proteins that exhibit antiviral activity 2. Virus infection signaled by TLRs & other PRRs, resulting interferon production 3. Constitutively expressed or induced by interferons 4. Often themselves antagonized by virus proteins (virulence factors) 5. Most often, cell-autonomous (act within the cells) 6. Exceptions are the interferons themselves: cytokines that act upon the interferon-producing cell (autocrine) and between cells (paracrine) 7. Cell intrinsic factors can be increased by interferons 8. Restriction factors target specific steps in the HIV-1 life cycle, but unlike antiretroviral drugs, they do so in a way that makes it difficult for HIV-1 to evolve resistance to the inhibitor through simple evasive mutations (maybe) 9. Intrinsic Cellular Defenses Are Determinants of Viral Host Range 10. Blocks to cross-species transmission are imposed by APOBEC3G and TRIM5 proteins that appear particularly powerful a. They are especially potent anti-viral proteins b. Constitutively expressed lentiviral target cells: T cells and macrophages APOBEC-3 • APOlipoprotein B Editing Catalytic subunit-like 3 (cytidine deaminases) • APOBEC3G (384 amino acids, 46.4 kDa) is the prototype antiretroviral cytidine deaminase • Expressed in virus infected cells and incorporated into progeny virions • Upon virion entry into a new target cell, APOBEC3G acts during reverse transcription, primarily during the synthesis of the negative sense DNA strand • Deaminates the C4 position of 2’-deoxycytidine producing 2’-deoxyuridine • APOBEC3G preferentially acts on the third cytosine of the sequence 5’-CCCA-3’ • Causes major disruption of the coding potential of the viral genome • Viral counter measures: Vif proteins bind to APOBEC3 and recruit proteosome degradation. SIVcpz Vif and SIVsmm Vif both active against HIV-1 and HIV-2
APOBEC3A, APOBEC3B, APOBEC3D, APOBEC3F, and APOBEC3H, Immunity Review
TRIM5α • TRIpartite Motif family of proteins • Targets the incoming viral capsid prior to reverse transcription • Consequence, capsid disruption and degradation • Acts to promote proteosome-mediated capsid degradation, but proteosomeImmunity inhibitors do not block anti-viral activity • SPRY domain highly variable in primates Review • Dictates the retroviruses that are restricted by a particular TRIM5 variant • Human TRIM5α largely ineffective against primate lentiviruses, but monkey TRIM5α very effective
A proposed model of TRIM5a activity suggests that TRIM5a forms a complementary three- dimensional lattice around the incoming capsid.
Figure 2. Structure and Antiretroviral Activity of Restriction Factors (A) Architecture of APOBEC3G. (B) Ribbon representation of the C-terminal CDA domain of APOBEC3G (PDB entry 3IR2) consisting of a five-stranded b-sheet core surrounded by six a-helices. The b2-sheet (shown in green) is distorted to various degrees in all reported NMR (PDB entries 2JYW, 2KBO, and 2KEM) and X-ray structures (PDB entries 3IQS and 3IR2), due to the differential hydration of residues in each structure. The three flexible loops near the CDA catalytic site (shown in red) contribute to substrate binding. The residues coordinating the zinc atom (black sphere), either directly or via a water molecule (blue sphere), are shown as a stick representation. (C) Architecture of TRIM5a. (D) Left: NMR structure (PDB entry 2ECV; residues 1 to 78) of the RING domain of human TRIM5a. The putative E2 enzyme-binding domain is shown in brown. The Figure 2. Structure and Antiretroviral Activity of Restriction Factors residues coordinating the zinc atom (black sphere) are shown as a stick representation. Right: NMR structure (PDB entry 2YRG; residues 86 to 131) of the B-box (A) Architecture of APOBEC3G. domain of human TRIM5a. A hydrophobic cluster of residues (shown in pink) and Arg 119 (shown in green) in particular are critical for higher-order oligomerization. (B) Ribbon representation of the C-terminal CDA domain of APOBEC3G (PDB entry 3IR2)(E) consisting A proposed model of a five-strandedof TRIM5a activity suggestsb-sheet that core TRIM5 surroundeda forms a complementary by six a-helices. three-dimensional lattice around the incoming capsid. The RING domain (green circles), the coiled-coil and B-box domains (black lines), and the SPRY domain (pink rectangles) are indicated. The b2-sheet (shown in green) is distorted to various degrees in all reported NMR (PDB entries(F) Architecture 2JYW, of Tetherin.2KBO, and 2KEM) and X-ray structures (PDB entries 3IQS and 3IR2), due to the differential hydration of residues in each structure. The three flexible(G) loops A model near for the possible CDA catalytic configurations site adopted (shown by tetherinin red) dimers contribute (PDB entry to substrate 2XG7) during virion tethering. Tetherin dimers might trap virions by the binding. The residues coordinating the zinc atom (black sphere), either directly or via a waterincorporation molecule of one pair (blue of anchors sphere), into theare viral shown envelope as (left a stick and center representation. panels). Alternatively, tethering might be achieved through the multimerization of tetherin molecules that are distributed between virion envelope and cell membrane (right panel). N and C represent the termini of tetherin. (C) Architecture of TRIM5a. (H) Architecture of SAMHD1. (D) Left: NMR structure (PDB entry 2ECV; residues 1 to 78) of the RING domain of human TRIM5(I) Ribbona representation. The putative of the E2 HD enzyme-binding domain of SAMHD1 (PDB domain entry 3U1N) is shown with an in expanded brown. view The of the active site. The residues coordinating the zinc atom (gray sphere), water molecule (blue sphere) and the phosphate ion are shown as a stick representation. residues coordinating the zinc atom (black sphere) are shown as a stick representation. Right:For (A), (C), NMR (F), and structure (H), domains (PDB and motifs entry critical 2YRG; for function residues are highlighted 86 to 131) in color of theand numbers B-box indicate the amino acid positions. Stars indicate catalytic site domain of human TRIM5a. A hydrophobic cluster of residues (shown in pink) and Arg 119 (shownresidues. in green) in particular are critical for higher-order oligomerization. (E) A proposed model of TRIM5a activity suggests that TRIM5a forms a complementary three-dimensional lattice around the incoming capsid. The RING domain (green circles), the coiled-coil and B-box domains (black lines), and the SPRY domain (pink402 Immunity rectangles)37, September are indicated. 21, 2012 ª2012 Elsevier Inc. (F) Architecture of Tetherin. (G) A model for the possible configurations adopted by tetherin dimers (PDB entry 2XG7) during virion tethering. Tetherin dimers might trap virions by the incorporation of one pair of anchors into the viral envelope (left and center panels). Alternatively, tethering might be achieved through the multimerization of tetherin molecules that are distributed between virion envelope and cell membrane (right panel). N and C represent the termini of tetherin. (H) Architecture of SAMHD1. (I) Ribbon representation of the HD domain of SAMHD1 (PDB entry 3U1N) with an expanded view of the active site. The residues coordinating the zinc atom (gray sphere), water molecule (blue sphere) and the phosphate ion are shown as a stick representation. For (A), (C), (F), and (H), domains and motifs critical for function are highlighted in color and numbers indicate the amino acid positions. Stars indicate catalytic site residues.
402 Immunity 37, September 21, 2012 ª2012 Elsevier Inc. Immunity
Review Genes encoding APOBEC3G and TRIM5α are under postive selection in human beings… They have among the highest dN/dS ratios of all human genes
What is an dN/dS ratio? Remember this selection pressure occurred in the distant past, long before HIV infected human beings
Figure 3. Evolution of Restriction Factor and Accessory Gene Function (A) Nef proteins of SIVs antagonize tetherin by interacting with the tetherin cytoplasmic tail. The diagram is a schematic representation of the genetic conflict between them. Colored figures indicate tetherin sequences in the cytoplasmic tail that are recognized by Nef and are hence rapidly evolving under positive selection. (B) Cumulative frequency distribution of dN/dS ratios for 12,404 human-chimpanzee orthologous gene pairs. Adapted from previously computed data (Chim- panzee Sequencing and Analysis Consortium, 2005). Positive selection (dN/dS > 1) and purifying selection (dN/dS < 1) are indicated by purple and orange arrows, respectively. The dN/dS value for each restriction factor is indicated by the dotted lines. The percentage of orthologous gene pairs with lower dN/dS ratios is indicated by the solid lines. (C) Evolution of Vpu and Nef function as primate lentiviruses were transmitted between species. See text for details. a population of host variants by different viral species or strains. Blocks to cross-species transmission are imposed by Thus, the mere presence or absence of signatures of positive APOBEC3G and TRIM5 proteins that appear particularly power- selection in a given gene cannot, by itself, be construed as diag- ful, perhaps because (1) these two restriction factors are espe- nostic of the presence or absence of a virus-host interaction. cially potent inhibitors, and (2) they are constitutively expressed Nevertheless, each of the four classes of HIV and SIV restriction in the natural target cells of primate lentiviruses. HIV-1 and SIV factors exhibits signatures of positive selection over at least Vif proteins are universally capable of antagonizing APOBEC3G a portion of its sequence, in at least some mammalian lineages. proteins in their natural hosts but are often impotent when con- fronted with APOBEC3G proteins from other primates (Mariani Intrinsic Cellular Defenses Are Determinants of Viral et al., 2003; Malim and Bieniasz, 2012). Indeed, the inability of Host Range many SIV Vif proteins to induce degradation of human Positive selection causes high interspecies protein sequence APOBEC3G might explain why many SIVs have not been found variability in restriction factors. Consequently, viral adaption to in humans. Notably, SIVCPZ Vif and SIVSMM Vif are both active antagonize or evade a particular restriction factor variant in one against human APOBEC3G (Gaddis et al., 2004), and both of host species can come at the cost of susceptibility to restriction these lineages have successfully colonized humans (as HIV-1 factor variants in another potential host. Thus, antagonistic and HIV-2, respectively). coevolution of virus and a particular host can reduce the proba- Although human TRIM5a is largely ineffective as an inhibitor of bility that an individual viral species can evade or antagonize the primate lentiviruses (Kratovac et al., 2008), TRIM5a variants array of defense mechanisms that confront it when the opportu- found in monkeys can be very potent inhibitors, and those found nity to colonize a new host species arises. in some species are capable of restricting an impressively
Immunity 37, September 21, 2012 ª2012 Elsevier Inc. 405 Does Variation in Restriction Factors Contribute to AIDS Susceptibility in Human Beings?
• Some variation in HIV/AIDS susceptibility in humans can be attributed to CCR5 (rare) and MHC polymorphisms (e.g., HLA B57) • Subpopulations of humans encode inactivating or destabilizing polymorphisms in genes encoding TRIM5α, APOBEC3B, APOBEC3H • Variation among APOBEC3H haplotypes in terms of antagonism by Vif. Polymorphic mutations are found in TRIM5a (H43Y) and APOBEC3G (H186R) that decrease activity • Actual resistance or sensitivity to HIV correlated with TRIM5 or APOBEC3 polymorphisms in human beings has not been consistently found! • Resistance due to polymorphisms in TRIM5 family members in macques HAS been shown • How can we use this information: inhibitors of Vif has been identified HIV 3EXUALLY 4RANSMITTED $ISEASES
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IT IS CLASSIlED AS A MODERATE COMPLEXITY TEST UNDER #,)! THER IT ENCOUNTERS THE ANTIHUMAN )G' CONTROL AND IF AN THER IT ENCOUNTERS THE ANTIHUMAN )G' CONTROL AND IF AN AND IS USUALLY PERFORMED IN A CLINICAL LABORATORY AND IS USUALLY PERFORMED IN A CLINICAL LABORATORY ADEQUATE SPECIMEN WAS ADDED A RED LINE APPEARS IN THE ADEQUATE SPECIMEN WAS ADDED A RED LINE APPEARS IN THE CONTROL LOCATION CONTROL LOCATION 4HE TEST RESULT SHOULD BE READ NO SOONER THAN MIN4HE TEST RESULT SHOULD BE READ NO SOONER THAN MIN 5NI 'OLD 2ECOMBIGEN5NI 'OLD 2ECOMBIGEN ()6 4EST ()6 4EST UTES AND NO LATER THAN MINUTES AFTER THE TEST DEVICE IS UTES AND NO LATER THAN MINUTES AFTER THE TEST DEVICE IS 4HE 4HE 5NI 'OLD 5NI 'OLD 2ECOMBIGEN 2ECOMBIGEN ()6 ()6 4EST 4EST RECEIVED RECEIVED &$! &$! INSERTED INSERTED INTO INTO THE THE DEVELOPER DEVELOPER VIAL VIAL ! ! 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IN FOR USE WITH VENIPUNCTURE AND lN VALID NEGATIVE TEST RESULT 4HE TEST IS INVALID AND SHOULD BE VALID NEGATIVE TEST RESULT 4HE TEST IS INVALID AND SHOULD BE GERSTICK WHOLE BLOOD SPECIMENS ;= 4HE DEVICE CONSISTS GERSTICK WHOLE BLOOD SPECIMENS ;= 4HE DEVICE CONSISTS REPEATED WITH A NEW DEVICE IF NO LINE APPEARS AT THE CONREPEATED WITH A NEW DEVICE IF NO LINE APPEARS AT THE CON OF A RECTANGULAR PLASTIC TEST CARTRIDGE AND A DROPPER BOTTLE OF A RECTANGULAR PLASTIC TEST CARTRIDGE AND A DROPPER BOTTLE TROL LOCATION OR IF LINES APPEAR OUTSIDE THE AREAS INDICATED TROL LOCATION OR IF LINES APPEAR OUTSIDE THE AREAS INDICATED OF OF BUFFER BUFFER SOLUTION SOLUTION &IG &IG 0EPTIDES 0EPTIDES FROM FROM THE THE IMMUNO IMMUNO BY THE TRIANGLES ;=BY THE TRIANGLES ;= DOMINANT REGION OF THE ()6 ENVELOPE ARE IMMOBILIZED DOMINANT REGION OF THE ()6 ENVELOPE ARE IMMOBILIZED $ESIGNED AS A POINT OF CARE ()6 TEST /RA1UICK$ESIGNED AS A POINT OF CARE ()6 TEST /RA1UICK HAS HAS ON A NITROCELLULOSE STRIP IN THE TEST REGION 2EAGENTS ARE ON A NITROCELLULOSE STRIP IN THE TEST REGION 2EAGENTS ARE BEEN BEEN USED USED IN IN NUMEROUS NUMEROUS SETTINGS SETTINGS INCLUDING INCLUDING LABOR LABOR AND AND ALSO BOUND AT THE CONTROL REGION TO INDICATE WHETHER THE ALSO BOUND AT THE CONTROL REGION TO INDICATE WHETHER THE DELIVERY ;s= AMBULATORY CLINICAL SITES ;= EMERGENCY DELIVERY ;s= AMBULATORY CLINICAL SITES ;= EMERGENCY TEST IS FUNCTIONING CORRECTLY BUT THESE DO NOT DETECT )G' TEST IS FUNCTIONING CORRECTLY BUT THESE DO NOT DETECT )G' DEPARTMENTS DEPARTMENTS ; = ; = HOSPITAL HOSPITAL INPATIENT INPATIENT SERVICES SERVICES ;= ;= AND THUS APPEARANCE OF THE CONTROL LINE DOES NOT VALIDATE AND THUS APPEARANCE OF THE CONTROL LINE DOES NOT VALIDATE 'REENWALD *, UNPUBLISHED DATA CORRECTIONAL FACILITIES 'REENWALD *, UNPUBLISHED DATA CORRECTIONAL FACILITIES THAT THAT ADEQUATE ADEQUATE PATIENT PATIENT SPECIMEN SPECIMEN HAS HAS BEEN BEEN ADDED ADDED /NE /NE ;= AND FOR OCCUPATIONAL EXPOSURES ;n= !DDITION;= AND FOR OCCUPATIONAL EXPOSURES ;n= !DDITION DROP OF SPECIMEN IS ADDED TO THE SPECIMEN WELL ON THE DROP OF SPECIMEN IS ADDED TO THE SPECIMEN WELL ON THE ALLY ALLY /RA1UICK /RA1UICK HAS HAS ALSO ALSO BEEN BEEN USED USED BY BY THE THE MILITARY MILITARY IN IN TEST CARTRIDGE FOLLOWED BY FOUR DROPS OF WASH BUFFER 4HE TEST CARTRIDGE FOLLOWED BY FOUR DROPS OF WASH BUFFER 4HE BATTLElELD OPERATIONS ;=BATTLElELD OPERATIONS ;= SPECIMEN SPECIMEN COMBINES COMBINES WITH WITH THE THE COLORIMETRIC COLORIMETRIC REAGENT REAGENT AND AND MIGRATES MIGRATES ALONG ALONG THE THE NITROCELLULOSE NITROCELLULOSE STRIP STRIP PAST PAST THE THE TEST TEST AND AND CONTROL CONTROL REGIONS REGIONS 4HE 4HE TEST TEST IS IS READ READ TO TO MINUTES MINUTES AFTER AFTER 2EVEAL2EVEAL© ' 2APID ()6 !NTIBODY 4EST © ' 2APID ()6 !NTIBODY 4EST SPECIMEN SPECIMEN IS IS ADDED ADDED ! ! LINE LINE IN IN BOTH BOTH THE THE TEST TEST AND AND CONTROL CONTROL /N !PRIL THE &$! APPROVED THE 2EVEAL/N !PRIL THE &$! APPROVED THE 2EVEAL© 2APID © 2APID REGIONS INDICATES A REACTIVE TEST A LINE IN ONLY THE CONTROL REGIONS INDICATES A REACTIVE TEST A LINE IN ONLY THE CONTROL ()6 !NTIBODY 4EST TO DETECT ()6 ANTIBODIES IN SERUM ()6 !NTIBODY 4EST TO DETECT ()6 ANTIBODIES IN SERUM REGION REGION INDICATES INDICATES A A NEGATIVE NEGATIVE TEST TEST 7HEN 7HEN USED USED WITH WITH WHOLE WHOLE OR PLASMA )N *UNE IT WAS SUPERSEDED BY THE SECOND OR PLASMA )N *UNE IT WAS SUPERSEDED BY THE SECOND BLOOD THE TEST IS VALID ONLY IF THE CONTROL LINE IS PRESENT BLOOD THE TEST IS VALID ONLY IF THE CONTROL LINE IS PRESENT GENERATION 2EVEALGENERATION 2EVEAL© ' TEST WHICH INCORPORATES AN INTER© ' TEST WHICH INCORPORATES AN INTER AND AND THE THE SAMPLE SAMPLE WELL WELL IS IS RED RED INDICATING INDICATING THAT THAT AN AN ADEQUATE ADEQUATE NAL NAL CONTROL CONTROL ;= ;= 2EVEAL 2EVEAL© © ' ' CONSISTS CONSISTS OF OF A A TEST TEST CARTRIDGE CARTRIDGE BLOOD SAMPLE HAS BEEN ADDED ;= BLOOD SAMPLE HAS BEEN ADDED ;=
Valid Results 4. Label each device with the appropriate patient information / ID. 5. Draw up adequate sample to the first gradation on the pipette using one of the disposable pipettes included in the kit. Use only the pipette included in the kit and do not reuse.
6. Holding the pipette vertically over the sample port, add one (1) free falling drop of sample carefully. Do not add the full volume contained within the pipette. REPORT AS REPORT AS Allow the sample to absorb into the PRELIMINARY POSITIVE NEGATIVE paper in the sample port. Ensure air Test line present No test line present bubbles are not introduced into the Control line present Control line present sample port. Discard the pipette in a Full red color at Sample Port Full red color at Sample Port biohazard waste container. Reactive Test Result Non-Reactive Test Result A pink/red line of any intensity appears in the A pink/red line of any intensity appears in the 7. Holding the dropper bottle of Wash device window adjacent to word "Test" AND a device window adjacent to word "Control" AND Solution in a vertical position, add four second pink/red line of any intensity appears a full red color appears in the Sample Port, but (4) drops of Wash Solution to the adjacent to word "Control" AND a full red color no pink/red line appears in the device window Sample Port. appears in the Sample Port. This indicates a adjacent to "Test". This indicates a Non- Reactive result that is interpreted as Reactive result that is interpreted as Negative Preliminary Positive for HIV-1 and/or HIV-2 for HIV-1 and/or HIV-2 antibodies. antibodies.
8. Set the timer for 10 minutes and start timing the test. TEST PROCEDURE SERUM, PLASMA AND CONTROLS; SERUM AND PLASMA SUITABLE FOR CLIA MODERATE SETTING ONLY 9. Read test results after 10 minutes but not more than 12 minutes incubation Test Procedure time. 1. Ensure that the Subject Information Leaflet has been given to the subject. PRINCIPLES OF THE PROCEDURE 2. Allow the kit (unopened devices and wash solution) to reach room temperature (15 – 27°C Uni-Gold™ Recombigen® HIV-1/2 was designed as a rapid immunoassay and is intended to / 59.0 – 80.6°F) (at least 20 minutes) if previously stored in the refrigerator. Once at room detect antibodies to HIV-1 and/or HIV-2 in human serum, plasma and whole blood10. (venipunctureRefer to the Test Results and Interpretation of Whole Blood Samples below. Note there is a temperature remove the required number of Uni-Gold™ Recombigen® HIV-1/2 devices and fingerstick). different interpretation for Whole Blood Samples from that for Plasma or Serum Samples. from their pouches. Perform no more than 10 tests at one time. ™ ® Uni-Gold Recombigen HIV-1/2 11. If testing whole blood check for full red color in sample port. The sample port must contain 3. Lay the devices on a clean flat surface. Uni-Gold™ Recombigen® HIV-1/2 uses proteins representing regions of the HIVred color virus. for test If to be valid. A pink/red line must appear adjacent to the word control. A antibodies to HIV-1 and/or HIV-2 are present in the sample, they combine with these proteinspink/red line and may appear adjacent to the word test. If no red color is seen in the sample port 4. Label each device with the appropriate patient information / ID. a color reagent and this complex binds to the proteins in the test forming a visible pink/redrepeat band test with in fresh device. 5. Draw up adequate sample to the 1206506 the test region of the device adjacent to the word ‘Test’. INTERPRETATION FOR WHOLE BLOOD SAMPLE first gradation on the Pipette using The control line should always appear as a visible pink/red band in the control region of the device Invalid Results one of the disposable pipettes to indicate that the test device is functioning correctly. A reactive result is indicatedFOR Aby TEST a pink/red TO BE VAL ID A CONTROL LINEPRINCIPLES MUST BE OF PRESENT THE PROCEDURE AND TH E SAMPLE PORT included in the kit. Use only the Disposable Pipette included in the band in the test region of the device. A non-reactive result occurs in the absenceMUST of CONTAIN detectable FULL Uni - GoldRED™ COLOUR Recombigen® HIV-1/2 was designed as a rapid immunoassay and is intended to Pour d'autres langues Para outras línguas kit and do not reuse. If Kit Controls detect antibodies to HIV-1 and/or HIV-2 in human serum, plasma and whole blood (venipuncture Für andere Sprachen Για τις άλλες λώσσες levels of antibodies to HIV-1 and/or HIV-2 in the specimen; consequently no visually detectable are being run, these must be used and fingerstick). Para otras lenguas För andra språk band develops in the test regionUni of the-Gold device™ .Recombigen ® HIV-1/2 as described in the package insert Per le altre lingue For andre språk Uni-Gold™ Recombigen® HIV-1/2 uses proteins representing regions of the HIV virus. If provided with the Kit Controls. MATERIALS PROVIDED antibodies to HIV-1 and/or HIV-2 are present in the sample, they combine with these proteins and Dla innych języków For andre sprog www.trinitybiotech.com a color reagent and this complex binds to the proteins in the test forming a visible pink/red band in 6. Holding the Disposable Pipette 1206506 the test region of the device adjacent to the word ‘Test’. vertically over the sample port, add The control line should always appear as a visible pink/red band in the control region of the device one (1) free falling drop of sample to indicate that the test device is functioning correctly. A reactive result is indicated by a pink/red carefully. Do not add the full Pour d'autres langues Para outras línguas band in the test region of the device. A non-reactive result occurs in the absence of detectable Read this package insert completely before using the product. Follow the directions carefully. Not Für andere Sprachen Για τις άλλες λώσσες levels of antibodies to HIV-1 and/or HIV-2 in the specimen; consequently no visually detectable volume contained within the Para otras lenguas För andra språk band develops in the test region of the device. Disposable Pipette Allow the doing so may result in incorrect test results. Before performing testing, all operators MUST read sample to absorb into the paper in Per le altre lingue For andre språk MATERIALS PROVIDED and become familiar with Universal Precautions for Prevention of Transmission of Human Dla innych języków For andre sprog www.trinitybiotech.com the sample port. Ensure air immunodeficiency Virus, Hepatitis B Virus and Other Blood-Borne Pathogens in Health-Care bubbles are not introduced into the REPORT AS REPORT AS REPORT AS REPORT AS REPORT AS sample port. Discard the Settings. INVALID INVALID INVALID INVALID INVALID Read this package insert completely before using the product. Follow the directions carefully. Not Disposable Pipette in a biohazard doing so may result in incorrect test results. Before performing testing, all operators MUSTTest read line No test line No test line No test line Test line waste container. and become familiar with Universal Precautions for Prevention of Transmission of pres Humanent present present present present CLIA COMPLEXITY; immunodeficiency Virus, Hepatitis B Virus and Other Blood-Borne Pathogens in Health-Care 7. Holding the dropper bottle of Wash Settings. No control line No control line Control line Control line Control line Solution in a vertical position, add present present present present present four (4) drops of Wash Solution to WAIVED FOR WHOLE BLOOD FINGERSTICK AND VENIPUNCTURE SAMPLES CLIA COMPLEXITY; Full red color at Full red color at No red color at Not full red No red color at the Sample Port Sample Port Sample Port Sample Port color at Sample Sample Port WAIVED FOR WHOLE BLOOD FINGERSTICK AND VENIPUNCTURE SAMPLES Port MODERATE COMPLEXITY FOR SERUM AND PLASMA SAMPLES MODERATE COMPLEXITY FOR SERUM AND PLASMA SAMPLES Each kit contains: No pink/red line No pink/red line Red color is not Red color is not Red color is not appears in the appears in the seen in the seen in full seen in the a) 20 Test Devices (individually pouched) Each kit contains: 8. Set the timer for 10 minutes and NAME AND INTENDED USE NAME AND INTENDED USE device window a)device 20 window Test Devices (individuallySample Port. pouched) sample well. Sample Port. start timing the test. b) Wash solution 5.0 ml adjacent to b)adjacent Wash to solution 5.0 ml White of sample Uni-Gold™ Recombigen® HIV-1/2 is a single use rapid immunoassay, for the qualitative detection Uni-Gold™ Recombigen® HIV-1/2 is a single use rapid immunoassay, for the qualitative detection word "Control" c)word 20"Control" Disposable Pipettes for use with serum,pad plasmaremains. or venipuncture whole blood. To be c) 20 Disposableof antibodies Pipettes to forHIV -1 use and/or with HIV - serum,2 in serum, plasma plasma and or wholevenipuncture blood (venipuncture whole and blood. To be used also with Controls (Catalog number 1206530) 9. Read test results after 10 minutes whether or not whether or not a of antibodies to HIV-1 and/or HIV-2 in serum, plasma and whole blood (venipuncture and used also withfingerstick). Controls Uni -Gold™(Catalog Recombigen® number HIV 1206530)-1/2 is intended for use in point of care settings as an d) 20 Disposable Fingerstick Sample Collection and Transfer Pipettes for use with fingerstick but not more than 12 minutes a pink/red line pink/red line fingerstick). Uni-Gold™ Recombigen® HIV-1/2 is intended for use in point of care settings as an aid in diagnosis of infection with HIV-1 and/or HIV-2. whole blood incubation time. d) 20 Disposable Fingerstick Sample Collection and Transfer Pipettes for useappears with infingerstick the appears in the e) 20 Subject Information Leaflets aid in diagnosis of infection with HIV-1 and/or HIV-2. whole blood This test is suitable for use in appropriate multi-test algorithms designed for the statisticaldevice window f)device 1 Packagewindow Insert validation of rapid HIV test results. e) 20 Subject Information Leaflets adjacent to adjacent to word "Test". word "Test". This test is suitable for use in appropriate multi-test algorithms designed for the statistical f) 1 Package Insert RESTRICTIONS Materials required and available as an accessory to the kit 10. Refer to the interpretation guide for serum and plasma. A pink/red line must appear Uni-Gold™ Recombigen® HIV Controls Kit. Catalog number 1206530. validation of rapid HIV test results. Sale of Uni-Gold™ Recombigen® HIV-1/2 is limited to clinical laboratories The test should The test should The test should The test should The test should adjacent to the word control. A pink/red line may appear adjacent to the word test. that have an adequate quality assurance program, including planned systematicbe repeated in beEach repeated pack of in Kit Controlsbe repeatedcontains; HIV in -1 Positivebe repeated Control, 1 invial (red cap),be repeated (0.5ml), HIV in -2 Positive Control, 1 vial (green cap), and Negative Control, 1 vial (black cap) (0.5ml) and a activities to provide adequate confidence that requirements for quality will beduplicate met. with duplicate with duplicate with duplicate with duplicate with RESTRICTIONS Materials required and available where thereas an is assuranceaccessory that operatorsto the willkit receive and use the instructional package insert. fresh devices. fresh devices. fresh devices. fresh devices. fresh devices. Sale of Uni-Gold™ Recombigen® HIV-1/2 is limited to clinical laboratories Uni-Gold™ Recombigen® materials.HIV Controls Kit. Catalog number 1206530. MATERIALS REQUIRED BUT NOT PROVIDED Uni-Gold™ Recombigen® HIV-1/2 is approved for use only by an agent of a clinical laboratory. Each pack of Kit Controls contains; HIV-1 Positive Control, 1 vial (red cap), (0.5ml), HIV-2 Timer or stopwatch that have an adequate quality assurance program, including planned systematic The test subjects must receive the "Subject Information Leaflet" prior to specimen collection, Blood collection devices, for testing of venipuncture whole blood, serum or plasma activities to provide adequate confidence that requirements for quality will be met. Positive Control, 1 andvial appropriate (green informationcap), and when Negative test results Control,are provided. 1 vial (black cap) (0.5ml) and a Biohazard disposal container Uni-Gold™ Recombigen® HIV-1/2 is not approved for use to screen donors of blood, plasma, Disposable gloves where there is assurance that operators will receive and use the instructional package insert. cells or tissues. materials. MATERIALS REQUIREDSUMMARY BUT NOT PROVIDED For Fingerstick samples the following additional material are required. Uni-Gold™ Recombigen® HIV-1/2 is approved for use only by an agent of a clinical laboratory. Adhesive bandages Timer or stopwatch HIV is the causative agent of AIDS (Acquired Immunodeficiency Syndrome). AIDS is the end stage The test subjects must receive the "Subject Information Leaflet" prior to specimen collection, of a drawn out process in which the immune system of an infected person and its ability to control Lancet capable of producing a 50μl droplet Blood collection devices,infections for or testing malignant of proliferativevenipuncture disorders whole are progressively blood, serum destroyed or .1plasma HIV is mainly Sterile wipes and sterile gauze pads and appropriate information when test results are provided. 1 Page 3 of 7 - EN Biohazard disposal containertransmitted by unprotected sexual intercourse or from mother to child. Most frequently, HIV WARNINGS Uni-Gold™ Recombigen® HIV-1/2 is not approved for use to screen donors of blood, plasma, infection is diagnosed by tests that assess whether an individual’s immune system has produced Disposable gloves an HIV-specific immune response (antibodies to HIV).1 For in vitro diagnostic use cells or tissues. In the USA the standard laboratory test algorithm (set of different tests) may take 48 hours to one Read the package insert completely before use. It is very important that the correct SUMMARY For Fingerstick samplesweek before the followingresults may be additional made availabl materiale. This algorithm are required.consists of screening with an enzyme procedure is followed. Not adding the patient sample may lead to a false negative result immunoassay (EIA) followed by confirmation by Western blot (WB) or immuno-fluorescent (IFA) (i.e. a missed positive). HIV is the causative agent of AIDS (Acquired Immunodeficiency Syndrome). AIDS is the end stage Adhesive bandagesmethods. Lancet capable of producing a 50μl droplet of a drawn out process in which the immune system of an infected person and its ability to control During the last 20 years, HIV infection and severe HIV-related diseases (e.g., AIDS) have become 1. Read the package insert completely before using the product. The instructions must infections or malignant proliferative disorders are progressively destroyed.1 HIV is mainly Sterile wipes anda sterileleading cause gauze of illness pads and death in the United States. Approximately 800,000-900,000 persons be followed carefully as not doing so may result in inaccurate results. in the United States are infected with HIV and approximately 275,000 of these persons might not 2. Before performing testing all operators must read and become familiar with the 1 transmitted by unprotected sexual intercourse or from mother to child. Most frequently, HIV know they are infected.2 WARNINGS Universal Precautions for Prevention of Transmission of Human Immunodeficiency Virus, Hepatitis B Virus and other Blood-Borne Pathogens in Health-Care settings.8 infection is diagnosed by tests that assess whether an individual’s immune system has produced 3. The FDA has approved this kit for use with serum, plasma and whole blood 1 Approximately 25 million persons each year in the United States are tested for HIV. Publicly an HIV-specific immune response (antibodies to HIV). For in vitro diagnosticfunded use counseling and testing programs conduct approximately 2.5 million of these tests each (venipuncture and fingerstick) specimens. Use of the kit with specimens other than year. In 1995, 25% of these individuals testing HIV positive and 33% of persons testing HIV those specifically approved for use with this device may result in inaccurate test negative at publicly funded clinics did not return for their test results. Rapid tests to detect HIV results. In the USA the standard laboratory test algorithm (set of different tests) may take 48 hours to one Read the packageantibody insert can be completelyperformed within 20 before minutes, use.enabling It health is - verycare providers important to supply thatdefinitive the correct4. This test kit is CLIA-waived for use only with fingerstick whole blood and week before results may be made available. This algorithm consists of screening with an enzyme negative and preliminary positive results to patients at the time of testing, potentially increasing the venipuncture whole blood samples. procedure is followed.overall effectiveness Not adding of counseling the patient and testing sample programs. may In comparison, lead to results a false from enzymenegat ive result5. Uni-Gold™ Recombigen® HIV-1/2 is for diagnostic use only and is not to be used for immunoassay (EIA) followed by confirmation by Western blot (WB) or immuno-fluorescent (IFA) (i.e. a missed positive).immunoassays (EIAs) currently used for HIV screening often are not available for 1-2 weeks.3 screening donors of blood, plasma, cells or tissues. methods. Using rapid tests, during 1995, a total of 697,495 more persons would have learned their HIV 6. Perform test at room temperature (15 – 27°C / 59.0 – 80.6°F). 3 status. PRECAUTIONS
During the last 20 years, HIV infection and severe HIV-related diseases (e.g., AIDS) have become 1. Read the packageMany advances insert have completely been made before in HIV/AIDS using prevention the product. and treatment, The including instructions the mustSafety Precautions a leading cause of illness and death in the United States. Approximately 800,000-900,000 persons be followed developmentcarefully of as effective not antiretroviraldoing so therapies may resultthat have in reduced inaccurate HIV related results. illness and death. 1. Standard precautions for handling infectious agents should be observed when using this Early knowledge of HIV infection is now recognized as a critical component in controlling the kit. 2. Before performing testing 2 all operators must read and become familiar with the in the United States are infected with HIV and approximately 275,000 of these persons might not spread of HIV infection. Rapid HIV testing allows clients to receive results the same day in a 2. Wear standard protective clothing such as a lab coat and disposable gloves when handling 2 Universal Precautionssingle visit, which foris useful Prevention in urgent medical of circumstancesTransmission and settings of wherHumane clients Immunodeficiency tend not to specimens and assay reagents in accordance with local regulations. know they are infected. 2 Virus, Hepatitisreturn B for Virus HIV test and results other (e.g., some Blood STD- clinics)Borne. Advances Pathogens in these in areas Health have resulted-Care insettings .8 3. Wash hands thoroughly after use. revised recommendations for HIV screening of pregnant women,4,5 treating opportunistic infections 4. In the case of Wash Solution contact with eyes, rinse immediately with plenty of water and Approximately 25 million persons each year in the United States are tested for HIV. Publicly 3. The FDA hasand other approved sexually transmitted this kit and bloodfor borne use diseases with and serum, managing plasma occupational and and non whole- blood seek medical advice. occupational exposures and prophylaxis.6,7 funded counseling and testing programs conduct approximately 2.5 million of these tests each (venipuncture and fingerstick) specimens. Use of the kit with specimens other thanAppropriate biosafety practices should be followed when handling specimens and year. In 1995, 25% of these individuals testing HIV positive and 33% of persons testing HIV those specifically approved for use with this device may result in inaccurate testreagents. These precautions include, but are not limited to, the following: negative at publicly funded clinics did not return for their test results. Rapid tests to detect HIV results. 4. This test kit is CLIA-waived for use only with fingerstick whole blood and antibody can be performed within 20 minutes, enabling health-care providers to supply definitive Page 1 of 7 - EN negative and preliminary positive results to patients at the time of testing, potentially increasing the venipuncture whole blood samples. overall effectiveness of counseling and testing programs. In comparison, results from enzyme 5. Uni-Gold™ Recombigen® HIV-1/2 is for diagnostic use only and is not to be used for immunoassays (EIAs) currently used for HIV screening often are not available for 1-2 weeks.3 screening donors of blood, plasma, cells or tissues. Using rapid tests, during 1995, a total of 697,495 more persons would have learned their HIV 6. Perform test at room temperature (15 – 27°C / 59.0 – 80.6°F). 3 status. PRECAUTIONS
Many advances have been made in HIV/AIDS prevention and treatment, including the Safety Precautions development of effective antiretroviral therapies that have reduced HIV related illness and death. 1. Standard precautions for handling infectious agents should be observed when using this Early knowledge of HIV infection is now recognized as a critical component in controlling the kit. 2 spread of HIV infection. Rapid HIV testing allows clients to receive results the same day in a 2. Wear standard protective clothing such as a lab coat and disposable gloves when handling single visit, which is useful in urgent medical circumstances and settings where clients tend not to specimens and assay reagents in accordance with local regulations. return for HIV test results (e.g., some STD clinics).2 Advances in these areas have resulted in 3. Wash hands thoroughly after use. revised recommendations for HIV screening of pregnant women,4,5 treating opportunistic infections 4. In the case of Wash Solution contact with eyes, rinse immediately with plenty of water and and other sexually transmitted and bloodborne diseases and managing occupational and non- seek medical advice. occupational exposures and prophylaxis.6,7 Appropriate biosafety practices should be followed when handling specimens and reagents. These precautions include, but are not limited to, the following:
Page 1 of 7 - EN HIV Western Blot
• Confirmatory test to exclude false-positive results • Nitrocellulose membrane is probed with patient serum • If the patient has been exposed to HIV, + bands will be observed on the Western blot Discussion points:
Who developed the first commercialized HIV test? What did it measure? What is being measured in an Elisa or in a Western blot assay? Why did it take three or four years to develop an assay? Is the assay perfect in diagnosing HIV infection? Why or Why not? Were there alternatives to measuring antibody specific to HIV in screening the blood supply? Did the blood banking industry try them? Which groups were in favor of screening donors, which groups were against?
Biosensors and Bioelectronics Volume 42 2013 69 - 75