Evoluzione genetica di HIV ed evoluzione clinica della malattia AIDS: due aspetti correlati?

1. Evoluzione genetica di HIV ed evoluzione clinica della malattia AIDS: due aspetti correlati? Carlo Federico Perno

2. Why does a Virus evolve? • A virus needs to evolve to: • Infect different cell types • Rapidly become resistant to otherwise highly effective antiviral drugs • Evade the immune system

3. Virus transmission Obstacles for virus transmission: Natural Host genetics Host Immune system Viral replication rate Etc Artificial Vaccines Passive immunity Antiviral drugs A continuous evolution allows viruses to achieve new characteristics able to overcome these obstacles, and be successful in their replication effort

4. Why does a Virus evolve? • A virus needs to evolve to: • Infect different cell types • Rapidly become resistant to otherwise highly effective antiviral drugs • Evade the immune system • …SURVIVE !!!

5. Consequences - The most fit virus, with the highest chances to survive, does not kill the host or, at minimum, kills the host in a long run - To be selected and expanded, it kills the host at a rate lower than other viruses of the same species - Ex: HIV subtype A vs Subtype D

6. Does HIV-1 genetic diversity have an effect on clinical progression? Subtype C vs. subtype A,P at log-rank = 0.2 Subtype D vs. subtypeA, P at log-rank = 0.05 Analysision 145 HIV-1 infected Kenyan women followed from the time of HIV-1 acquisition. “HIV-1 Subtype Dis associated with faster disease progression than Subtype A in spiteof similar plasma HIV-1 loads” Baetan JM , JID 2007

7. How does a virus evolve? Evolution = genetic variation • The steps in virus evolution are: • generation of diversitythroughmutation, recombination,andgenome segment reassortmentin multipartite genomes • competitionamong the generated variants • selectionof those mutants showing the largest phenotypic advantage in a given environment

8. FUNDAMENTALS OF MOLECULAR EVOLUTION

9. “All the organic beings which have ever lived on this earth have descended from some one primordial form” • Charles Darwin • From this idea, each characteristic of a species could be the result of a peculiar evolutionary history: • Peacock’s ancestors • The number and the sequences of his genes • The catalytic ability of his enzymes • His needs • The structure of his cells • His environmental fitness • ... This is his evolutionary history • Evolution is the unifying theory of biology “Nothing in biology makes sense except in the light of the evolution” Theodosius Dobzhansky

10. In biology a mutationis a randomly derived change to the nucleotide sequence of the genetic material of an organism. • Non lethal mutations accumulate within the gene pool and increase the amount of genetic variation. The abundance of some genetic changes within the gene pool can be reduced by natural selection, while other "more favorable" mutations may accumulate and result in adaptive evolutionary changes.

13. Does occurrence of mutations mean their necessary selection and appearance (fixation) in circulating virus strains? NO

14. Same amino acid, same protein Silent Mutation Synonymous Point Mutations Substitution of a Nucleotide Early stop of protein Mutation Non Sense A C G T 4 nucleotides X X X 1 codon = 1a.a. 43 = 64 codons -> 20 a.a. Mutation NON synonymous Different amino acid, protein mutated

15. Objective To examine tropism after ART failure using a clinically validated genotypic tropism assay in a large sample of treatment-experienced patients HIV-1 tropism was assessed at baseline and virologic failure over 48 weeks in patients receiving an optimized background regimen (OBT) with a maraviroc placebo (PBO) in the MOTIVATE 1 and 2 studies1 What is the relation between the HIV tropism and its evolution 15 1Gulick et al. N Engl J Med. 2008;359:1429-1441 Svicher et al. 2009

16. 16 Rate of successful V3 sequencing V3 sequencing was successful for 87 (80.5%) out of 108 samples 83 out of 87 have also the trofile result available. For the remaining 4, Trofile failed to assess viral tropism. All of them resulted R5 with Trofile at screening Svicher et al. 2009

17. ~90% of patients with R5 tropism at screening had an R5 result at treatment failure. 17 Viral tropism has been predicted by Geno2Pheno algorithm at a false positive rate of 5% using 87 V3 sequences *P< 0.001

18. 18 Analysis of 65 patients where R5-usage is maintained both at screening and at failure Svicher et al. 2009

19. Despite tropism stability (R5 both at screening and at failure), the majority (23/35) of V3 positions shows higher entropy at failure than at screening suggesting viral evolution despite tropism stability 19 Change in entropy from screening to failure V3 positions The analysis was performed in the sub set of 65 patients with R5-tropism at baseline and failure (using geno2pheno at FPR of10%). The Shannon entropy was calculated for each V3 position following the formula: H(i) = - Σ P(si) log P(si) (where s=A,S,L,… for the 20 amino acids Ala, Ser, Leu, . . .). The difference between entropy at screening and at failure at each V3 position is reported in the graph. Svicher et al. 2009

20. 20 Increased genetic diversity between screening and failure significantly correlates with higher duration of treatment Rho is the Spearman's rank correlation coefficient. Rho, ranging from -1.00 to 1.00, is a measure of the strength and direction of the association between two variables. A positive coefficient indicates that the variables X and Y increase in a correlated manner. The genetic distance (mean number of substitutions per site) of V3 sequences from screening to failure for each patient and treatment duration were used to calculate Rho. The analysis was performed in the sub-set of 65 patients with R5-tropism at baseline and failure (using geno2pheno algorithm at FPR of 10%). Svicher et al. 2009

21. Despite the high natural genetic variability of V3, the frequency of tropism switches remains limited 21 - An accumulation of synonymous substitutions was observed from screening to failure in all 87 patients (100%) - An accumulation of non-synonymous (amino acidic) substitutions was observed in 26/87 patients (28.9%) - Tropism switches were observed in only 4 patients (3 from R5 to X4, 1 from X4 to R5) [4/87, 4.6%] The analysis was performed on all 87 patients on study (using geno2pheno algorithm at FPR of 5%). Average observation from baseline to virological failure was 150 days

22. Switches from R5 to X4 usage is mainly driven by a shift of viral species R5 at screening X4 at failure R5 at screening X4 at failure R5 at screening X4 at failure The analysis was performed on the 3 samples resulting R5 at screening and X4 at failure using geno2pheno algorithm at both 5% and 10%. Genetic distance is the mean number of substitutions per site. The high genetic distance values and the high number of amino acid substitutions from screening to failure support the shift from an R5-using strain to an X4-using strain Svicher et al. 2009

23. CONSEQUENCES C. If a mutation with lower fitness remains fixed, we obtain a minority species (called quasispecies), that may become predominant if the environment changes Ex 1. Antiviral pressure that selects for a viral strain with lower sensitivity to drugs 2. Immunological pressure by a vaccine that selects for an escape mutant not neutralized by the immune system In the case of viruses, this switch in predominance may take days (not millennia!!) - Selection of strains resistant to antiviral drugs

24. Re-emergence of the most fit R5-virus Re-Emergence of R5!! Tropism at Failure: D/M Baseline Tropism: Designated R5 Maraviroc in Suboptimal Regimen Stop Maraviroc X4 HIV Not Detected at <4% R5 X4 D/M Non-functional clone Lewis M, et al. 16th IHIVDRW, 2007. Abstract 56.

25. CDC stage: C3 Clinical Case: Id 186 - Patient infected with HIV-1 B subtype Age: 46 Sex: M Risk Factor: Not known GRT during therapy interruption GRT under antiretroviral treatment GRT March ’02 (ARV: 3TC d4T ABC LPV/r) PR: L10I M36V M46L I54V L63P A71T V82A N88D L90M I93L RT: M41L E44E/D D67N L74L/V V118V/I M184V G190G/E/Q/R L210W T215Y K219K/N G333E GRT September ‘02 PR: L63P V77I I93L RT: G333E GRT March ’05 (ARV: 3Tc d4T LPV/r) PR: L10I K20R L33F M36M/I/V M46I I54V L63P A71T G73G/A V82A N88D L90M I93L RT: M41L E44E/D D67D/N V118I M184V L210W T215Y G333E GRT May ‘06 PR: L63P V77I I93L RT: G333E GRT January ’08 (ARV: AZT 3TC ABC DRV/r) PR: L10I K20R V32I L33F M36I K43T M46I I47V I54V L63P A71T G73A/T I84V N88D L90M I93L RT: M41L E44D D67N V118I M184V L210W T215Y G333E

26. Virus under drug pressure selects, among thousands of quasispecies present in the body, the virus strain with the greatest fitness in that environment Wild type strain (the most fit) without drugs Highly mutated (resistant) strain in the presence of drugs No chances of winning the battle until viral replication is sharply decreased/nullified

27. CONSEQUENCES • The replication is a necessary prerequisite for occurrence and appearance of mutations • Without replication, no mutation By decreasing the replication rate of a virus, we dramatically decrease its ability to escape immune system and antiviral drugs

28. BUT…… ……If a mutation produces a variant with low fitness, and/or this mutation is not fixed, this new variant disappears Ex. Loss of viral species

29. Population Dynamics 1 fixed mutation polymorphism maintained Allele Frequency lost mutation 0 Time

30. Effective sample size: the genetic bottleneck Population dynamics of alleles Each different symbol represent a different allele. A mutation event in the sixth generation gives rise to a new allele. The figure illustrates fixation and loss of alleles during a bottleneck event, and the concept of coalescence time (tracking back the time to the most recent common ancestor of the gray individuals). N: population size. Coalescence time Bottleneck event Mutation event Adapted from The Phylogenetic Handbook 2009, M Salemi and AM Vandamme

31. PRACTICAL CONSEQUENCE • By reducing the sample size of a species (bacteria, viruses, etc) we dramatically reduce the chance that the species mutates and thus escapes pressure by chemotherapy and/or immune system: - Success of antivirals and antibiotics despite a small remaining number of microorganisms - Success of vaccines against viruses with low mutational rate - Insuccess of vaccines against highly mutating viruses - Insuccess of vaccines targeted against genes with high rate of mutations

32. - The case of smallpox virus- The case of influenza virus

33. Evolutionary abilities of Variola Variola has a single linear double stranded DNA genome of 186 kilobase pairs. Some studies showed the presence of a low mutation rate. A similar situation is present in other component of orthopoxviruses genus. Number of SNPs found in different couples of viral isolates Adapted form Li et al., 2007 *Single Nucleotide Polymorphisms Variola is lacking of great evolutionary potential

34. Smallpox Vaccine Its history is strictly connected to the birth of modern vaccinology. Variolation 1796: Edward Jenner. 1977: last case of smallpox.

35. Such good result was due to… the biological characteristics of the organism, vaccine technology, surveillance and laboratory identification, effective delivery of vaccination programmes and international commitment to eradication. Smallpox virus has no host reservoir outside humans!!.

36. The case of influenza virus

37. The evolutionary power of antigenic shift The last known flu pandemics

38. Genesis of “Swine flu” H1N1 virus Avian flu virus (unknown subtype) Human H3N2 flu virus Swine H1N1 flu virus H1N1 “Swine flu” virus Classical swine flu virus H1N1 H3N2 swine virus

39. The reservoir hosts act as variability source for the new evolutionary steps of flu virus

40. The evolutionary novelty of “Swine flu” virus … gene sequences collected from the USA for swine flu (subtype H1N1) in the year 2009 are evolutionarily widely different form the past few years sequences…the 2009 sequences are evolutionarily more similar to the most ancient sequence reported in the NCBI database collected in 1918. (Sinha et al., 2009)

41. CONSEQUENCES Smallpox: Low rate of polymerase errors + lack of animal reservoir (even in the presence of high replication rate) = Eradication possible (and obtained indeed!!) Flu: High rate of polymerase errors + presence of multiple animal reservoirs (+ high rate of recombination) = Eradication impossible New vaccine required every year

42. Resistance to anti-HIV drugs is the most elegant, and practically relevant, example of the consequences of viral evolution

43. What about the effect of resistance on clinical outcomes?

44. HIV-1: Drug resistance development It’s important to detect resistant quasispecies before the treatment starting or as soon as possible during treatment Toxicity No Adherence Bioavailability Patients’ Metabolism Reservoir

45. Percentage of patients’ viruses who had RTI resistance mutations at t0 and of those who acquired such mutations from t0 to t1 by specific mutation/drug class RTI resistance at t0 RTI resistance from t0 to t1 In patients kept on the same virologically failing cART regimen for a median of 6 months, there was considerable accumulation of drug resistance mutations Cozzi-Lepri et al., AIDS 2007

46. Poor survival in drug-class multi-resistance 0 DCMR 1 DCMR 2 DCMR P at log-rank <0.001 3 DCMR Zaccarelli, AIDS 2005

47. Main Findings In multivariable analyses, patients with drug resistance mutations to ≥ 2 classes during first 2 years of HAART at significantly higher risk of AIDS progression or death Cozzi-Lepri A, et al. AIDS. 2008;22:2187-2198.

48. Virus continues to evolve if kept under pressure of failing antiviral therapy. This may increase cross-resistance, and then decrease chances of efficacy of subsequent drugs and regimens. In the frame of a correct therapeutic sequencing, first failing therapies should be changed as soon as possible after definition of virological failure.

49. Conclusions • Viruses represent the best model of evolution on the earth • They mutate in days faster than what humans have ever changed in millennia • Their evolution capacity is function of several factors • The host represents the most important extrinsic factor • Through a proper use of interdisciplinary tools (mathematics, physics, biochemistry, molecular biology, biology, pharmacy, medicine) we can reasonably predict their evolution, and define ways and consequences of the interaction with humans

50. Conclusion (II) • The understanding of viral evolution has major consequences in medicine, of key practical relevance: • Identification of targets for viral vaccines • Definition of potential outcomes of massive vaccinations • Eradication, infection containment, functional cure of infected people • Setting therapeutic strategies against viral infections • Definition of the chances of success of antivirals (resistance testing, antivirograms) • Select therapies with the greatest chances of success (Ex. multiple drugs against viruses with high mutation rate)