Non: Week 3
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Purpose
The purpose of this week's lab is to look at a study characterizing the rates of CD4 T cell decline in HIV patients by their evolution in the unique host environments.
New Terminology
- CD4 - a CD marker that occurs on T‐helper cells and is involved in MHC class II restricted interactions (Carmack et al, 2006)
- viral load - The number of viral particles (usually hIV) in a sample of blood plasma. HIV viral load is increasingly employed as a surrogate marker for disease progression. It is measured by pCR and bDNA tests and is expressed in number of HIV copies or equivalents per millilitre. (Biology Online Dictionary)
- seroconversion - "The stage in an immune response when antibodies to the infecting agent are first detected in the bloodstream" (Martin & Hines, 2008)
- peripheral blood mononuclear cells (PBMC) - "The mononuclear cells of the blood: monocytes and lymphocytes" (Lackie, 2019)
- monocytes - "A circulating mononuclear phagocyte. Monocytes differentiate into macrophages having emigrated into tissues." (Lackie, 2019)
- lymphocytes "White blood cells of the lymphoid series. There are two main classes, T and B lymphocytes (T cells and B cells); the former subdivided into subsets (helper, suppressor, cytotoxic T cells) and variously involved in cell-mediated immunity and stimulating B lymphocytes. B cells, when activated, secrete antibody." (Lackie, 2019)
- reverse-transcription-PCR (RT-PCR) - "used for amplifying molecules of RNA, by initially converting the RNA into its complementary DNA molecule using the enzyme reverse transcriptase and then following the standard procedure for PCR. It can be used to analyse gene expression in samples taken from certain tissues or under particular conditions." (Hine, 2019)
- coreceptor - A protein on the surface of a cell that serves as a second binding site for a virus or other molecule (AidsInfo NIH)
- nested PCR - A PCR that is done to increase sensitivity and specificity; involves two sequential amplification reactions, each of which uses a different pair of primers (Green and Sambrook, 2019)
- substitution rate - expected number of mutations that will fix in the population per unit time (Lehmann, 2014)
- epitope - the part of an antigenic molecule to which the t-cell receptor responds, a site on a large molecule against which an antibody will be produced and to which it will bind (Biology Online Dictionary)
- escape variant - a viral variant that can escape immune detection (Frederich et. al, 2014)
Outline
Abstract
- sample size: 15 injection drug users, selected for differences in CD4 T cell decline
- patients with rapid/moderate disease progression - favored nonsynonymous mutations
- patients with low progression/"nonprogressors" - selected against nonsynonymous mutations
- CD4 T cell declines mirror accumulation of mutations, types of mutations most favorable to host
Introduction
- HIV-1 has high mutation, replication rates
- in stable environment = leads to selection for "best fit" virus
- in unstable environment = variability in genetic effects
- could lead to overwhelming diverse minority populations that overload immune system
- studying the patterns of evolution helps elucidate the factors that viruses can adapt to
- previous studies had small sample, did not examine sequence patterns, limited time points
- study found "higher levels of genetic diversity is most frequently associated with more rapid CD4 T cell decline"
Methods
- AIDS Linked to Intravenous Experiences: Baltimore, MD
- 15 subjects (to combat limited sample size in other studies)
- monitored subjects every 6 months for 4 years (to combat limited time points)
- three classifications for the study
- rapid progressors - <200 CD4 T cells within 2 yrs.
- moderate progressors - 200-650 CD4 T cells during 4 yr. period
- nonprogressors - >650 CD4 T cells through 4 yr. period
- nested PCR amplfied 285bp region of env gene of peripheral blood mononuclear cells (PBMC)
- studies had shown the viral RNA is integrated into the DNA here, unstable and only persists for few days, closely related to viral RNA
- nested PCR product cloned into pUC19 plasmid
- sequenced using Sanger chain termination method
- phylogenetic trees generated using MEGA with the neighbor-joining algorithm, Tamura-Nei distance measuring
- correlation analysis was done to compare changes in CD4 T cell count between after a year with genetic diversity and mutational divergence
- dS/dN ratio was calculated after determining whether each difference was synonymous or nonsynonymous
- subjects 9 and 15 was further examined using phylogeny as a result of their high initial genetic variation
- regression line comparing rate of change of divergence with diversity
Results
- CD4 T cell decline was variable: median ranged from +53 to -594/year
- viral genomic RNA ranged from 1702 to 321,443 copies/mL
- genetic sequence analysis on hypervariable V3 region of env
- important site of host-virus interaction, able to tolerate frequent mutations
- 873 clones sequenced, analyzed
- rate of change in median diversity ranged from -2.94nt to 5.10nt/clone/year
- rate of divergence from 0.13% to 2.09% of nts/clone/year
- most of the subjects (13/15) had homogeneous viruses at initial visit; the other 2 had seroconversion up to 7 months prior to initial visit
- diversity and divergence increased over time for the three categories
- rapid progressor group had much significantly (P<0.001) rate of increase in divergence than nonprogressor group; not significantly (P=0.017) higher than moderate progressor group
- differences in slopes was significant between rapid and nonprogressors (P=0.001); not significant between rapid and moderate (P=0.08)
- both divergence and diversity were significantly negatively correlated with CD4 T cell count after a year = greater genetic diversity/divergence at a given visit --> likely greater CD4 T cell decline after a year
- ratio of the (rate of synonymous mutations per potential site of synonymous mutation) to the (rate of nonsynonymous mutations per potential site of nonsynonymous mutation) dS/dN
- rapid and moderate progressors had a median ratio of 0.4 favoring NS mutations
- nonprogressors had a median ratio value of 1.6, selecting against NS mutations
- no selective advantage for viruses carrying changes in envelope protein structure
- dS values were not significantly different among all the groups
- most phylogenetic trees (10/15) showed no predominance of single strain for an extended period of time
Figures/Tables
- Fig. 1 - plots CD4 T cell count, diversity and divergence for all 15 subjects over time (4 years)
- shows the marked differences in T cell decline between the different classifications
- Table 1 - summarizes data gathered from the subjects; divided into respective classifications; includes raw CD4 count, number of time points, viral copy number, rate of CD4 decline, nt differences, slope of nt change, slope of nt mutations from consensus (aka divergence), and median dS/dN
- assigns numerical values to the differences between the groups with rapid progressors having higher annual rates of CD4 T cell decline, steeper slopes in nt differences and divergence as well as having lower median dS/dN
- Fig. 2 - box and whisker plot comparing the slopes of diversity and divergence in table 1
- Fig. 3 - a phylogenetic tree from one subject showing the immense number of clones and mutations in a given subject
- Fig. 4 - phylogenetic trees of four subjects highlighting the sporadic and non-linear evolution of each of their HIV variants
Discussion
- higher levels of genetic diversity, divergence lead to increased CD4 T cell loss
- does not follow the idea of a best fit virus
- other studies have not necessarily shown similar results
- McDonald et al. had similar results in that greater genetic divergence was seen in rapid producers; however, diversity was lower for rapid progressors than slow progressors
- explained by fact that different time periods were measured; also less time points taken
- Wolinsky et al. showed less genetic diversity in fast progressors
- may be an "exceptional subject who failed to develop any effective immune response" (Markham, et al. 1998)
- Nowak results mirrored this study's results with regard to genetic diversity and divergence
- progression exhibited a frequency-dependent selection or independent evolution of variants from different geographical/environmental sites
- McDonald et al. had similar results in that greater genetic divergence was seen in rapid producers; however, diversity was lower for rapid progressors than slow progressors
- a hypothesis for favoring nonsynonymous mutations as seen in nonprogressors is to avoid overly replication-competent viruses that would be targeted by immune system
Analysis of Paper
- supported their hypothesis well with a good amount of relevant data from many perspectives
- convincing hypothesis but likely needs more experimentation
- would like an even larger sample size with more time points to better characterize T cell changes
- highly interesting how a more unstable virus actually benefited long-term survival rather than a stable one as we typically see in evolution of normal organisms
Scientific Conclusion
Overall, the paper highlighted the finding that HIV-1 selects for a more unstable, constantly changing virus through genetic diversity and genetic divergence. While it is not exactly clear why this is, the scientists believe this is due to frequency dependent selection; the host immune system targets the most common phenotypes present so the virus favors a constantly changing phenotype to avoid detection.
Acknowledgements
- My partners this week, Carolyn and Drew, worked together for this journal club to present Figure 1 of the paper.
- I used and modified this week's assignment protocol.
"Except for what is noted above, this individual journal entry was completed by me and not copied from another source."
Non (talk) 21:10, 5 February 2020 (PST)
References
- Coreceptor Definition. (n.d.). Retrieved February 5, 2020, from https://aidsinfo.nih.gov/understanding-hiv-aids/glossary/173/coreceptor#
- Epitope. (2014, May 12). Retrieved February 5, 2020, from https://www.biology-online.org/dictionary/Epitope
- Friedrich, T. C., Dodds, E. J., Yant, L. J., Vojnov, L., Rudersdorf, R., Cullen, C., ... & Sette, A. (2004). Reversion of CTL escape–variant immunodeficiency viruses in vivo. Nature medicine, 10(3), 275-281.
- Green, M. R., & Sambrook, J. (2019). Nested polymerase chain reaction (PCR). Cold Spring Harbor Protocols, 2019(2), pdb-prot095182.
- Markham, R. B., Wang, W. C., Weisstein, A. E., Wang, Z., Munoz, A., Templeton, A., ... & Yu, X. F. (1998). Patterns of HIV-1 evolution in individuals with differing rates of CD4 T cell decline. Proceedings of the National Academy of Sciences, 95(21), 12568-12573. doi: 10.1073/pnas.95.21.12568
- Martin, E., & Hine, R. (2008). seroconversion. In A Dictionary of Biology. : Oxford University Press. Retrieved 30 Jan. 2020, from https://www.oxfordreference.com/view/10.1093/acref/9780199204625.001.0001/acref-9780199204625-e-6465.
- Lackie, J. PBMC. In Nation, B. (Ed.), A Dictionary of Biomedicine. : Oxford University Press. Retrieved 30 Jan. 2020, from https://www.oxfordreference.com/view/10.1093/acref/9780191829116.001.0001/acref-9780191829116-e-7154.
- Lackie, J. monocyte. In Nation, B. (Ed.), A Dictionary of Biomedicine. : Oxford University Press. Retrieved 30 Jan. 2020, from https://www.oxfordreference.com/view/10.1093/acref/9780191829116.001.0001/acref-9780191829116-e-6162.
- Lackie, J. lymphocytes. In Nation, B. (Ed.), A Dictionary of Biomedicine. : Oxford University Press. Retrieved 30 Jan. 2020, from https://www.oxfordreference.com/view/10.1093/acref/9780191829116.001.0001/acref-9780191829116-e-5619.
- Lehmann L. (2014). Stochastic demography and the neutral substitution rate in class-structured populations. Genetics, 197(1), 351–360. doi:10.1534/genetics.114.163345
- OpenWetWare. (2020). BIOL368/S20:Week 3. Retrieved February 5, 2020, from https://openwetware.org/wiki/BIOL368/S20:Week_3
- Viral load. (2014, May 12). Retrieved February 5, 2020, from https://www.biology-online.org/dictionary/Viral_load
- (2006). CD4. In Cammack, R., Atwood, T., Campbell, P., Parish, H., Smith, A., Vella, F., & Stirling, J. (Eds.), Oxford Dictionary of Biochemistry and Molecular Biology. : Oxford University Press. Retrieved 5 Feb. 2020, from https://www.oxfordreference.com/view/10.1093/acref/9780198529170.001.0001/acref-9780198529170-e-3064.
- (2019). polymerase chain reaction. In Hine, R. (Ed.), A Dictionary of Biology. : Oxford University Press. Retrieved 30 Jan. 2020, from https://www.oxfordreference.com/view/10.1093/acref/9780198821489.001.0001/acref-9780198821489-e-3522.
