Predicting the Onset of AIDS Robert Arnold, Alex Cardenas, Zeb Russo LMU Biology Department 10/5/2011

Outline What causes a subject to develop AIDS from HIV and what separates AIDS from HIV? Focusing on dS/dN ratio The definition of AIDS, the subjects affected, and their similarities, ALIVE information New hypothesis involving the division of subjects into those with AIDS, trending towards AIDS, and AIDS free trending away Research comparison, proving assumptions incorrect Further comparisons between the subjects with AIDS and those without Comparing our results with our paper

dS/dN ratio related to AIDS development determined that low dS/dN ratios, subjects that select either for nonsynonymous mutation or not against it were the subjects to develop AIDS The subjects picked were 4, 9, 11, and 14, all with 0.0 dS/dN ratios along with subject 10 with a 0.2 and subject 1 with a 0.3

Outline What causes a subject to develop AIDS from HIV and what separates AIDS from HIV? Focusing on dS/dN ratio The definition of AIDS, the subjects affected, and their similarities, ALIVE information New hypothesis involving the division of subjects into those with AIDS, trending towards AIDS, and AIDS free trending away Research comparison, proving assumptions incorrect Further comparisons between the subjects with AIDS and those without Comparing our results with our paper

AIDS and CD4 counts CDC definition of AIDS is a CD4 count below 200 Once diagnosed, cannot be reversed Makes our first hypothesis irrelevant since all ‘rapid progressors’ drop below 200, AKA all 6 have AIDS Subjects 1, 3, 4, 10, 11, 15

Outline What causes a subject to develop AIDS from HIV and what separates AIDS from HIV? Focusing on dS/dN ratio The definition of AIDS, the subjects affected, and their similarities, ALIVE information New hypothesis involving the division of subjects into those with AIDS, trending towards AIDS, and AIDS free trending away Research comparison, proving assumptions incorrect Further comparisons between the subjects with AIDS and those without

Revised hypothesis separating those with AIDS from others Separated into 3 categories Those with AIDS: 1, 3, 4, 10, 11, 15 Those trending to AIDS: 7, 8, 9, 14 Those free of and trending away from AIDS: 2, 5, 6, 12, 13 New vision; which subjects developed AIDS? Began to focus on ALIVE research to go beyond Markham’s 4 year period

Development of two new questions Since we can tell who has AIDS, we would now like to determine whether there are any similar clones of the env gene across the AIDS subjects Does a median ds/dn ratio below 1.0 or lower determine whether you will get AIDS or not?

Outline What causes a subject to develop AIDS from HIV and what separates AIDS from HIV? Focusing on dS/dN ratio The definition of AIDS, the subjects affected, and their similarities, ALIVE information New hypothesis involving the division of subjects into those with AIDS, trending towards AIDS, and AIDS free trending away Research comparison, proving assumptions incorrect Further comparisons between the subjects with AIDS and those without Comparing our results with our paper

Our division of the Patients

Random clonal comparison To determine whether there were any similarities between clones of those who developed AIDS during the study and those at risk, we performed a ClustalW on a random selection of two clones from each subject

2 Clones Rooted Tree

Comparison of dS/dN Subject No. of observations CD4 Median intravisit nucleotide differences among clones Virus copy number (×103) Annual rate of CD4 T cell decline Slope of change in intravisit nucleotide differences per clone per year Slope of divergence (% nucleotides mutated from baseline consensus sequence per year) Median dS/dN AIDS   Subject 4 4 1,028 0.9 6.8 −593 4.64 2.09 Subject 10 5 833 1.71 99.3 −363 3.16 1 0.2 Subject 11 753 2.27 62.2 1.11 0.32 Subject 15 707 15.16 171 −362 −2.94 0.68 0.7 Subject 3 819 1.82 302.5 −294 0.53 0.74 Subject 1 3 464 5.64 307.6 −117 5.1 1.55 0.3 At Risk Subject 7 1,072 317.6 −392 −0.79 1.35 1.3 Subject 8 7 538 1.24 209 −92 1.68 1.16 0.5 Subject 9 8 489 9.49 265 −11 1.58 1.21 Subject 14 9 523 50.9 −51 1.69 0.6 Not at Risk Subject 2 715 1.64 21.6 30 1.32 0.49 1.8 Subject 5 749 2.5 260.6 −41 0.06 1.4 Subject 6 405 2.82 321.4 52 1.92 0.82 0.4 Subject 12 6 772 2.8 44 0.62 0.13 Subject 13 671 0.87 1.7 53 0.28 3.5

Neither Assumption is Definitive Using the original data from the Bedrock website, we determined who actually developed AIDS over the full study 1, 3, 4, 6, 7, 8, 9, 10, 11, 14, 15 Only 2, 5, 12 and 13 avoided the progression to AIDS over the course of the study

Outline What causes a subject to develop AIDS from HIV and what separates AIDS from HIV? Focusing on dS/dN ratio The definition of AIDS, the subjects affected, and their similarities, ALIVE information New hypothesis involving the division of subjects into those with AIDS, trending towards AIDS, and AIDS free trending away Research comparison, proving assumptions incorrect Further comparisons between the subjects with AIDS and those without Comparing our results with our paper

2 Clones Rooted Tree Redux

Comparison of dS/dN Subject No. of observations CD4 Median intravisit nucleotide differences among clones Virus copy number (×103) Annual rate of CD4 T cell decline Slope of change in intravisit nucleotide differences per clone per year Slope of divergence (% nucleotides mutated from baseline consensus sequence per year) Median dS/dN AIDS   Subject 4 4 1,028 0.9 6.8 −593 4.64 2.09 Subject 10 5 833 1.71 99.3 −363 3.16 1 0.2 Subject 11 753 2.27 62.2 1.11 0.32 Subject 15 707 15.16 171 −362 −2.94 0.68 0.7 Subject 3 819 1.82 302.5 −294 0.53 0.74 Subject 1 3 464 5.64 307.6 −117 5.1 1.55 0.3 At Risk Subject 7 1,072 317.6 −392 −0.79 1.35 1.3 Subject 8 7 538 1.24 209 −92 1.68 1.16 0.5 Subject 9 8 489 9.49 265 −11 1.58 1.21 Subject 14 9 523 50.9 −51 1.69 0.6 Not at Risk Subject 2 715 1.64 21.6 30 1.32 0.49 1.8 Subject 5 749 2.5 260.6 −41 0.06 1.4 Subject 6 405 2.82 321.4 52 1.92 0.82 0.4 Subject 12 6 772 2.8 44 0.62 0.13 Subject 13 671 0.87 1.7 53 0.28 3.5

Immune Relaxation Hypothesis Evolutionary rates slow due to disrupting immune function In relation to our claims, the virus infects specific CD4+ T cell count. The immune system is the positive selecting agent in C2-V5 region of env. With progression to AIDS, the immune effectors and the virus can alter the course of evolution. Lower dS/dN ratios were observed in a single patient CD4+ T Cells disrupted by infection respond to epitopes coded by env and in driving env sequence evolution.

Divergence Non-synonymous divergence stabilizes Seems to coincide with disease progression Synonymous divergence does not seem to stabilize Phylogenetic methods can’t be used per say in this paper No recombination is generally assumed And studies show that recombination rate in HIV populations in vivo is high

For our studies, < 1 indicated AIDs and > 1 indicated no AIDs Ratios dN/dS ratios > 1 indicates widespread adaptive evolution Most common among data sets, indicative of positive selection < 1 some sites are selectively constrained For our studies, < 1 indicated AIDs and > 1 indicated no AIDs

CD4+ T Cells In direct correlation with distinguishing AIDs Subjects < 300 AIDs Related it back to < 200 from previous graphs and tables. “Feedback loop” in play. Virus impairs HIV specific responses

Findings 11/15  AIDs CD4 T Cell Count dN/dS ratio New ?’s

Citations Markham RB, et al. 1998. Patterns of HIV-1 evolution in individuals with differing rates of CD4 T cell decline. Proc. Natl. Acad. Sci. USA 95: 12568-12573. Williamson S, et al. 2004. A Statistical Characterization of Consistent Patterns of Human Immunodeficiency Virus Evolution Within Infected Patients. Molecular Biology of Evolution, 22(3):456-468. Retrieved off of Web of Science September 28th, 2011.