# Presentation: 144686

## HIV/AIDS in 2004*

**	****	****	****	****	****      ****2003****	****Cumulative**
**Number of AIDS deaths:**
**	****North America****	****	****     20,000****	****   500,000**
**	****World****	****	****	****	****3,000,000****	****22,00,000**
**Total number of infected people:****	**
**	****North America****	****	****     940,000****	****  1,200,000**
**	****World****	****	****	****    ****	****40,000,000****	****62,000,000**
**Number of new infections:**
**	****North America****	****	****     45,000**
**	****World****	****	****	****	****5,000,000**
**Number of people cured of HIV****	****	****	****	****<1**
**Number of infections prevented by vaccination****	****	****<1**

**HIV/AIDS in 2004***

***UNAIDS, December, 2003**

## Slide 2

## Important Properties of HIV

**Infection requires CD4 protein on the surface of the cell as receptor.**
**Therefore can only infect CD4+ (“helper”) T cells and a few others.**
**Almost all infected cells die within a day or two after infection.**
**Infected CD4 cells make enough virus particles to infect about the same number of new cells (10-100 million). **
**Therefore, the infection in an individual persists by constant, repeated cycles of infection and cell death (about 1 a day).**
**These properties are also found in the benign SIV-monkey infections, but in humans there is a slow loss of total CD4 cells, leading eventually to failure of the immune system.**

## Slide 4

## RNA Copies/ml

**RNA Copies/ml**

**Appearance of 3TC-Resistant Mutations in Treated Patients**

## Slide 6

## Virus load (copies/ml)

**Virus load (copies/ml)**

**8**

**7**

**6**

**5**

**4**

**3**

**2**

**1**

**0**

**Years**

## Persistence of Cells latently Infected with HIV after Suppression of Viremia to “Undetectable” Levels

**Zhang et al NEJM 340:1605-1613**

**Persistence of Cells latently Infected with HIV after Suppression of Viremia to “Undetectable” Levels**

## Viremia Persists after Suppression by Antiretroviral Therapy 

**0               200             400             600            800**

**Time (days)**

**    ****10****7**

**    ****10****6**

**    ****10****5**

**    ****10****4**

**    ****10****3**

**    ****10****2**

**    ****10****1**

**    ****10****0**

**   ****10****-1**

**0              200            400            600            800**

**Time (days)**

**Plasma HIV-1 RNA (copies/ml)**

**Viremia Persists after Suppression by Antiretroviral Therapy**** **

**Notes:**
As shown here in twwo typical examples patients initiating ARV therapy with a standard regimen initially experience a rapid then somehat deceelerated declinbe in HIV RNA until viremia is no longer detectable using commercial assays

As John Mentioned the single copy and bDNA assays have excellent concordance  and quantitate the rapid ad slow phase declines in viremia. 

In contrast however, SCA continues to detect HIV throughout the treatment period of nearly 2 years
Of note in both of these patients a stable level of viremia was achieved that was distinct for each patient. As you can see,  Viremia remained quite stable; the  error bars for these determinations are approximated by the points themselves; Regression analysis revealed no additional evidence of decay with these regression lines are not significantly differenc from zero.

## HIV-Host Interaction

**1.  After early primary infection, HIV gives lifelong persistent infection leading to AIDS after about 10 years (on average).**
**2.  Persistence is due to constant replication of the virus and killing of 10****7****-10****9**** infected CD4+ T cells  at about 1 cycle/day.**
**3.  Smaller fractions of “latently infected” cells that  live much longer after infection are probably unimportant for the natural history of the infection, but very important for foiling treatment.**
**4.  Constant replication day after day, year after year, leads to extensive genetic variation.**
**	****• ****Antigenic escape.**
**	****• ****Drug resistance.**
**	****• ****Variation in coreceptor usage. **
**5.  The system remains in an extraordinarily robust quasi steady state for thousands of replication cycles before progressing to disease.**
**6.  We still don’t know how HIV causes AIDS. **

**HIV-Host Interaction**

## HIV Drug Resistance

**Introduction.**
**Mechanism of resistance.**
**Evolution of resistance.**

## The HIV Replication Cycle

**The HIV Replication Cycle**

**Adsorption to CD4 receptor **

**Entry via fusion**
**Following coreceptor binding**

**Reverse transcription**

**Transcription**

**Integration**

**Tat**

**Rev**

**splicing**

**Nef**

**Gag-Pro-Pol**

**Maturation**

**Vpu**

**Vif**

**Assembly**
**Budding**

## Anti-HIV Therapies

**Unsuccessful (so far):**
**	****• ****Immunotherapy.**
**	****• ****Gene therapy.**
**	****• ****Recombinant antiviral proteins.**
**	****• ****Herbal extracts.**
**	****• ****Faith healing.**
**Successful:**
**	****• ****Nucleoside RT inhibitors. (AZT, 3TC, ddI, ddC, d4T, etc.)**
**	****• ****Non nucleoside RT inhibitors. (Nevirapine, Efavirenz,     ****	****Delavirdine,etc.)**
**	****• ****Protease inhibitors. (Indinavir, Saquinavir, Nelfinavir, ****	****Ritonavir, etc.)**
**	****• ****Fusion inhibitors (Enfuvirtide).**
**Promising:**
**	****	****• ****Integrase inhibitors.**
**	****	****• ****Coreceptor inhibitors.**

**Anti-HIV Therapies**

## Approved anti-HIV Drugs2003

**Nonnucleoside RT Inhibitors:**

**Nucleoside RT**
**Inhibitors: **

**Zidovudine (AZT)**

**Zalcitabine (ddC)**

**Nelfinavir**
**Ritonavir**
**Saquinavir**

**Stavudine (d4T)**
**Tenofovir**

**Lopinavir**

**Nevirapine**

**Lamivudine (3TC)**

**Indinavir**

**Efavirenz**

**Didanosine (ddI)**

**Amprenavir**

**Delavirdine**

**Abacavir**

**Approved anti-HIV Drugs****2003**

**Fusion Inhibitors:**

**Enfuvirtide**
**(T20)**

## Deaths per 100 person-years

## HIV Drug Resistance

**1.  Occurs with all (effective) antivirals tested to date, *****in vivo***** and *****in vitro.  (“If you don’t get resistance, the drug’s no good.”)***
**2.  Is the most important factor preventing successful long term treatment.  (If resistance did not arise, we would probably not be here today.)**
**3.   Monotherapy almost always rapidly fails, most likely due to selection of mutants already present in the virus population.**
**4.   Our only way to deal with this problem at present is to throw enough drugs at it so that no preexisting variant is resistant to all of them, and that replication is sufficiently suppressed to prevent further evolution. **
**5.  A patient for whom this therapy has failed has very few treatment options left.**

**HIV Drug Resistance**

## Location of Drug Resistant Mutations in HIV Proteins

**0**

**100**

**200**

**300**

**400**

**500**

**Nucleoside RT inhibitors**

**Nonnucleoside RT inhibitors**

**Pyrophosphate RT inhibitors**

**Protease inhibitors**

**Binding/fusion inhibitors**

** ****A.  Reverse transcriptase**

** ****B.  Protease**

**D.  Env**

**Fingers**

**Palm**

**Fingers**

**Palm**

**Thumb**

**Connection**

**RNase H**

**Integrase inhibitors**

** ****C.  Integrase**

**gp120SU**

**gp41TM**

**600**

**700**

**800**

**900**

**V1**

**V2**

**V3**

**V4**

**V5**

**C1**

**C2**

**C3**

**C4**

**Ectodomain**

**Cytoplasmic domain**

**Amino Acid Position**

## Drug-Resistant Mutations in HIV

**NNRTI’s**
**3TC**
**AZT**
**Protease inhibitors**

## p66

- thumb

- fingers

- palm

- RNase H

- p51

- _pol active site_

- _RNase H _
- _active site_

**Courtesy of E. Arnold**

- _template_

- _primer_

## Locations of Drug Resistance Mutation

**Locations of Drug Resistance Mutation**
**Sites in HIV-1 RT/DNA Structure**

**Nucleoside drug resistance mutation sites**

**Non-nucleoside drug resistance mutation sites**

## NNRTI Resistance

**Occurs rapidly in patients and cell culture.**
**Virtually complete cross-resistance to chemically very different compounds.**
**Mutations in binding “pocket” at base of thumb domain.**
**Pocket does not exist in the native structure.**

## Binding of NNRTIs to HIV-1 RT

**Binding of NNRTIs to HIV-1 RT**

**Courtesy of E. Arnold**

**Notes:**
1. Now we focus at NNRTI binding pocket region.  
2. Shown here is a superposition of three potent NNRTIs (TIBO in green, a-APA in yellow, and nevirapine in purple). These are from the crystal structures of individual inhibitors complexed with HIV-1 RT.  
3. Despite of their chemical diversity, they bind to RT more or less in a similar fashion which can be assumed as a butterfly sitting on the b6- sheet. However, the second generation NNRTIs bind to HIV-1 RT at the same pocket but in very different modes.
4. Shown amino-acid residues either involve in the binding of an NNRTI or mutated to confer drug  resistance or do both.

## NNRTI binding pocket region in wild-type HIV-1 RT structure

**Leu 100**

**Lys 101**

**Lys 102**

**Tyr 188**

**Tyr 181**

**Lys 103**

**Glu 138 (B)**

**OW**

**NNRTI binding pocket region in wild-type HIV-1 RT structure**

****

****

****

**p51**

**Courtesy of E. Arnold**

**Notes:**
 Here we focus our attention at the sites of mutations (i.e. Tyr181 and Lys103) in the wild-type epo-enzyme structure.
 Tyr181 stacks with Glu138 of p66 where as the side-chain for Lsy103 is pointing outward.

## Inhibitor-protein Interactions

**Interactions are predominantly hydrophobic (with side-chains of L100, Y181, Y188, F227, W229, L234, and Y318).  **

**Inhibitor-protein Interactions**

**Courtesy of E. Arnold**

**Notes:**
 Shown here is the binding of 8-Cl TIBO to HIV-1 RT.
The Inhibitor-protein interactions for NNRTIs are predominantly hydrophobic .....
Our sites of mutations (Y181 and K103) are shown in red.
The aromatic side chain of Tyr181 interacts with the bound NNRTIs. 
Whereas, the side-chain of Lys103 has no direct interaction with the bound NNRTI.

## NNRTI binding pocket region in K103N/Y181C mutant structure

**Cys 181**

**Tyr 188**

**Glu 138 (B)**

**Asn 136 (B)**

**Asn 103**

**Lys 102**

**Lys 101**

**Leu 100**

**OW**

**NNRTI binding pocket region in K103N/Y181C mutant structure**

****

****

****

- 2.8 Å

- 3.1 Å

- 3.3 Å

**Courtesy of E. Arnold**

**Notes:**
 shown here is the NNRTI binding pocket region in the double-mutant (i.e. K103N and Y181C) HIV-1 RT structure determined at 2.5 A resolution.
 A consequence of K103N mutation is a H-bond between the side-chains of Asn103 and Tyr188 which was not present in the wild-type HIV-1 RT structure.  
 If we recall the structures of wild-tpe HIV-1 RT, Tyr188 flips away upon the binding of an NNRTI.  So this H-bond is an additional constrain that need to be broken for an NNRTI to bind.
 Glu138 loses interactions with the aromatic ring and changes its side-chain torsion at C-beta by about 120 degrees.
As a consequence, Glu138 develops a hydrogen bond with the S-gamma of Cys181 and another with Asp136 side-chain. 
5.  In summery, the double mutant epo-enzyme structure showed some important rearrangements in the pocket residues, particularly at the putative entrance.  Those changes may effect the process of NNRTI binding.  

## 3TC Resistance

**Occurs rapidly in patients and cell culture.**
**Mutations nearly always at M184 in active site.**
**RT from resistant virus is resistant to 3TCTTP incorporation.**
**Resistance due to steric hindrance.**

## Structure of 3TC 

**Structure of 3TC **

**Deoxycytidine**

**3TC**

- (-)-2 ́, 3 ́-dideoxy-3 ́-thiacytidine (3TC)

## Steric Hindrance in 3TC Resistance

- 3TC

- Sarafianos, et al.,1999.
- PNAS USA 96: 10027-10032.

**Steric Hindrance in 3TC Resistance**

## AZT Resistance

**Occurs rapidly in patients and cell culture.**
**Mutations not at active site or dNTP binding site.**
**RT from resistant virus not resistant to AZTTP incorporation.**
**Resistance due to excision (pyrophosphorolysis).**

## Courtesy of S. Hughes

**Courtesy of S. Hughes**

## Courtesy of S. Hughes

**Courtesy of S. Hughes**

## Clash Between Incorporated AZT and Active site Aspartic Acid Prevents Translocation 

- Clash Between Incorporated AZT and Active site Aspartic Acid Prevents Translocation

**Courtesy of S. Hughes**

## Other           NRTI

**Other           NRTI**

**AZT**

**Courtesy of S. Hughes**

## Protease Inhibitor Resistance

**Occurs rapidly in patients and cell culture.**
**Usually associated with multiple mutations that arise sequentially.**
**Initial (primary) mutations at or near active site, and often greatly reduce fitness.**
**Subsequent mutations often far away from active site (or even in *****gag*****), and, in many cases, act to improve fitness.**

## Courtesy of A. Wlodawer

**Courtesy of A. Wlodawer**

## Evolution of Protease Inhibitor Resistance in Vivo

**Time (weeks)**

**Relative virus load**

**Primary**

**mutation**

**Secondary**

**mutations**

**Evolution of Protease Inhibitor Resistance *****in Vivo***

## Modeling HIV Variation

**How do drug resistance mutations arise?**

## Evolution of 3TC Resistance

**Time, Weeks**

**Virus Load**

**Evolution of 3TC Resistance**

## Evolution of 3TC Resistance

**M184 (AUG)**

**M184I (AUG-****AUA****)**

**Time, Weeks**

**Virus Load**

**Evolution of 3TC Resistance**

## Evolution of 3TC Resistance

**M184 (AUG)**

**M184I (AUG-****AUA****)**

**M184V (AUG-GUG)**

**Time, Weeks**

**Virus Load**

**Evolution of 3TC Resistance**

## Evolution of HIV in Infected People

**Thesis:**
**1.  Drug-resistant mutations are present in the virus population  prior to therapy.**
**2.  In the absence of drug, these mutations are slightly deleterious to the virus.**

## Factors of HIV Evolution

**Factors of HIV Evolution**

**1.  Mutation**

## Factors of HIV Evolution

**Factors of HIV Evolution**

**More fit**

**Less fit**

**2. Selection**

## Factors of HIV Evolution

**Factors of HIV Evolution**

**More important in small populations of cells**

**Mutant proportion changes due to random sampling**

**3. Drift**

## Factors of HIV Evolution

**Factors of HIV Evolution**

**Etc.**

**Can be reversed by recombination or compensating mutations.**

**4. Linkage**

## Accumulation of Drug Resistant 

**Mutant frequency**

**.**

**0**

**3**

**.**

**0**

**2**

**.**

**0**

**1**

**1**

**/**

******

***N***

***s***

**1**

**/**

***s***

**0**

**Accumulation of Drug Resistant **
**Mutations Before Start of Therapy**

**0**

**0.01**

**0.02**

**0.03**

**Time, generations**
**(years after infection)**

**0**

**1000**

**2000**

**3000**

**(0)**

**(5)**

**(10)**

**(15)**

**Population size:**

## Accumulation of Drug Resistant 

**Mutant frequency**

**.**

**0**

**3**

**.**

**0**

**2**

**.**

**0**

**1**

**1**

**/**

******

***N***

***s***

**1**

**/**

***s***

**0**

**Accumulation of Drug Resistant **
**Mutations Before Start of Therapy**

**0**

**0.01**

**0.02**

**0.03**

**Time, generations**
**(years after infection)**

**0**

**1000**

**2000**

**3000**

**(0)**

**(5)**

**(10)**

**(15)**

**Population size:**

## Genetics of HIV-1 Populations in Infected Individuals

***National Cancer Institute at Frederick***

**HIV Drug Resistance Program**

## Objectives

**To obtain a detailed analysis of HIV-1 genetic diversity in infected patients to understand:**
** ****Roles of mutation and selection.**
** ****Replicating population size.**
** ****Extent of recombination.**
** ****Presence of compensatory mutations.**
** **

**Objectives**

## Patients in Study

**1. Recent HIV infection (n=3)**
- 10-100 days post-infection
- VL=12,000->500,000 copies/ml
**2. Chronic untreated HIV infection (n=3)**
- >4 mos-10 years
- VL=7,300-30,000 copies/ml
**3. Chronic HIV infection initiating ART (n=3)**
- >6 mos-15 years
- VL=86,000-1,500,000 copies/ml

## Limiting Dilution PCR Sequencing

***AKA: single genome sequencing (SGS)***

**Sequence   **

## Assay Characteristics

**• ****Amplicon includes nearly all major sites for ****	****	****	****resistance mutations **
**• ****Variation less than *****env*****, but gives adequate ****	****	****	****phylogenetic signal**
**• ****Does not give alignment problems**
**• ****Background:**
**	****error rate = 0.012%  **
**	****recombination rate < 1 per 60,000 nucleotides **

## Recent 

**Nucleotide Substitutions, %**

**0**

**5.5**

**2**

**4**

- HXB2 clippedp6prrt

- 0706-14

- 0706-15

- 0706-16

- 0706-17

- 0706-2

- 0706-10

- NG-5

- NG-7

- NG-6

- NG-10

- NG-2

- NG-11

- SR-4

- SR-5

- SR-3

- SR-2

- SR-6

- SR-11

- BP-16

- BP-3

- BP-4

- BP-7

**HXB2**

**Recent **
**HIV Infection**

**Patient 3**

**Typical**
**Chronic**
**Patient**

**Patient 2**

**Patient 1**

## Patients in Study

**1.  Recent HIV infection (n=3)**
**10-100 days post-infection**
**VL=12,000->500,000 copies/ml**
- 2.  Chronic untreated HIV infection (n=3)
- >4 mos-10 years
- VL=7,300-30,000 copies/ml
- 3.  Chronic HIV infection initiating ART (n=3)
- >6 mos-15 years
- VL=86,000-1,500,000 copies/ml

## Accumulation of Mutations Early after Infection

**0**

**.025**

**.050**

**.075**

**.100**

**.125**

**.150**

**.175**

**Days after infection**

**Mutation frequency (%)**

**.200**

**0**

**10**

**20**

**30**

**40**

**50**

**60**

**70**

## Conclusions:

**Recently Infected Patients**

** ****Virus populations were nearly homogenous**
** ****Mutation frequencies for most patients were not  distinguishable from the error  rate of the limiting dilution assay and well below those expected for neutral accumulation.**
** ****Strong purifying selection on HIV-1 populations early during HIV infection?**

## Patients in Study

- 1.  Recent HIV infection (n=3)
- 10-100 days post-infection
- VL=12,000->500,000 copies/ml
**2.  Chronic untreated HIV infection (n=3)**
**>4 mos-10 years**
**VL=7,300-30,000 copies/ml**
- 3.  Chronic HIV infection initiating ART (n=3)
- >6 mos-15 years
- VL=86,000-1,500,000 copies/ml

## HIV Diversity over Time in Chronic Infection

**Months**

**Viral Load**

**Patient 1**
**42 y.o. male**
**HIV infection>10 y**
**CD4=348 cells/μl**

**HIV Diversity over Time in Chronic Infection**

**0       4       8      12     16    20**

**10****6**

**10****6**

**10****4**

**10****2**

**10****0**

**No discernable change in tree over nearly 2 years.**

## Antiretroviral Therapy Initiated

- 3.1

- 0

**Start **
**Therapy**

- Patient 3
- 45 yo male
- HIV infection >15 y
- CD4=111 cells/l

**Similar HIV variants present **
**after >2 log decrease in viral load**

-        0      2      4     6      8     10     12

- Weeks

- 10

- 103

- 105

- 107

- Virus load

## Conclusions: Chronic Untreated Infection 

- •  **Diverse population: no two sequences are the same with the average intrapatient diversity of nearly 2%.**
**• ****No evident shift in population structure over 18 months.**
**• ****Extensive recombination. **
**• ****Evidence for compensatory mutations.**
**• ****Implies a large, genetically stable population.**

## Overall Conclusions: 

**  **
**3. These results are consistent with a very large population, replicating at a genetic balance, and subject to strong purifying selection, implying that resistance may be predictable.  **

**Overall Conclusions: **