Courtney L. Merriam Week 8

Purpose

The purpose of this experiment was to prepare for Week 9 Journal Club both individually and as a group.Individually, make an outline and list of definitions of the journal article. As a group, prepare a PowerPoint presentation and present it next week in class.

Methods and Results

Definitions of Terms

1. Nabs
   • An antibody that is capable of keeping an infectious agent, usually a virus, from infecting a cell by neutralizing or inhibiting its biological effect, for example by blocking the receptors on the cell or the virus. ¹
2. Epitopes
   • That 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. See also agretope. ⁴
3. Autologous
   • Derived from an organism’s own tissues or DNA. cf. heterologous, homologous. ⁴
4. Heterologous
   • Derived from the tissues or DNA of a different species. ⁴
5. Bootstrap
   • A statistical method for testing the reliability of a dataset. It involves creating a set of pseudo replicate datasets by re-sampling and is extensively used, for example, in phylogenetic analyses. ⁴
6. Amplicons
   • The DNA product of a polymerase chain reaction ⁴
7. Cotransfecting
   • The simultaneous transfection with two separate, unrelated nucleic acid molecules, one of which may contain a gene that is easily assayed and acts as a marker. ³
8. Infectivity
   • Refers to the ability of a microorganism to invade and replicate in a host tissue, whether the microbe is pathogenic or not. ⁶
9. Pseudoviruses
   • Virus-like particle composed of a viral coat protein enclosing an unrelated DNA sequence. Pseudo viruses are potentially useful as a means of delivering DNA into cells for therapeutic purposes or to induce antigen production and thus act as a vaccine. ⁴
10. Serological
    • The scientific study or diagnostic examination of blood serum, especially with regard to the response of the immune system to pathogens or introduced substances. ⁵

Outline

Introduction

• Goal is finding cross-reactive Nabs to help synthesize vaccines
• HIV-1 typed into nine lineages
• Type C accounts for 50% of worldwide HIV infections
  • Study focuses on analyzing subtype C
  • Especially dominant in India and Southern Africa
• Studying autologous and heterologous may bring improvements in immunogen design
  • CD4 epitopes may contain insights for neutralizing HIV-1-positive sera
• Highly variable
• Limitation: previous studies reliance on PCR involved env cloning
  • Can bring artificial recombination and resampling
  • Minor env changes in sequence can result in missed information
  • SGA and PCR methods utilized to study possible neutralization signatures that are linked with cross-reactive Nab responses associated with C type viruses

Results

• 34 of the 37 subjects infected with subtype C virus
  • three extra subjects infected with either D, A/C Recombinant, or G/J Recombinant
  • 13 of the C subtype had similar or exactly the same env sequences
  • 474 env genes were observed with only 377 being functional
• some recombinant genes gain either an advantage or they may become inert or lethal
  • pseudo virus infectivity linked to viral load but not env expression in each subject
• Neutralization signature analysis
  • Four signature sites identified
• three in gp120
• one in gp41
  • Possible one or more is false positive
  • Envs related to cross-reactive Nabs often mutated towards an Asn in position 413
  • Data leads to assumption v4 loop linked with neutralizing antibodies against subtype C

Discussion

• Analyzed several HIV-1 env genes in all 37 subjects
  • Used SGA and pPCR methods
• Sequence and functional analysis showed consistent clonal expansions
• Positive link between viral load and infectivity of env pseudo viruses
  • Highly variable env infectivity
  • signature amino acids in v4 region
  • implications for effective vaccines
• 13 of 37 individuals carried similar clusters of related sequences
  • nearly identical or identical
• beginning to diverge

Methods and Materials

• Plasma samples collected from 37 HIV-1 positive test subjects
  • No subjects treated with antiretroviral drugs
  • Subjects likely infected through heterosexual transmission
• Total of 474 env genes extracted from subjects
• pPCR and SGA performed for subjects
  • Phylogenetic tree constructed with complete env sequences
• Neighbor-joining method
• 16 plasma samples used for neutralization assay
  • 60 env pseudotypes

Figures and Tables

• Table 1
  • Graph of 36 subjects with summary of functional env genes and single genome analyses
• Figure 1
  • A phylogenetic tree showing the prevalent grouping of C subtype viruses as well as the D, A/C recombinant, and G/J recombinant subtypes
• Table 2
  • Table showing clonal expansion of env sequences in the test subjects, including percentage of total viral population, and varying amino acid numbers in env sequences
• Figure 2
  • Phylogenetic tree of only clonal expansion for env gene sequences for individuals who experienced env gene clonal expansion.
• Figure 3
  • Graph examining env pseudo virus activity. Comparing luciferase activity for each individual subject.
• Figure 4
  • Graph comparing viral load in terms of copies per mL and luciferase activity in order to find correlation
• Figure 5.
  • Western blot of HIV-1 proteins expressed in transfected cells, examining three of the 37 test subjects, visualized with secondary antibodies. Infectivity shown as a relative light unit.
• Figure 6
  • Phylogenetic tree of env sequences from 15 HIV-1 infected individuals. Color coded such that black was monophyletic, and red and blue used to show different clusters
• Figure 7
  • Neutralization graph showing the hierarchical clustering of viruses and sera. Sera clusters are organized according to neutralization ability.
• Figure 8
  • Amino acid sequences associated with neutralization capability in plasma. H means high neutralization activity for heterogeneous viruses, L has weak neutralizing ability.
• Figure 9
  • A computer rendered image of the crystal structure ligated gp120 with accompanying amino acids
• Figure 10
  • Comparison of neutralizing activity between low and high heterologous neutralizing plasma samples.

Data and Files

Journal Week 9 Presentation

Week 8 Kirchherr Reading ²

Conclusion

This week’s lab was more presentation oriented than anything else. It’s been fun developing the PowerPoint presentation and learning in depth the information I need to know for the graph and table presentations. The graphs and tables in the journal articles I’ve read have always either facilitated my understanding of the material, or further confused me when I couldn’t fully grasp the information being displayed. Taking the time to break down the tables and graphs in depth one by one has made me more confident that I can do it with any table or graph in the future

Acknowledgments

I collaborated with Avery Vernon-Moore, Shivum A Desai, Matthew R Allegretti, and Kam D. Dahlquist in class on this assignment. While I worked with the people noted above, this individual journal entry was completed by me and not copied from another source.

• Courtney L. Merriam 15:54, 18 October 2016 (EDT):

References

1 Biology Online Dictionary. (n.d.). Retrieved October 18, 2016, from http://www.biology-online.org/dictionary

2 Kirchherr, J. L., Hamilton, J., Lu, X., Gnanakaran, S., Muldoon, M., Daniels, M., ... & Musonda, R. M. (2011). Identification of amino acid substitutions associated with neutralization phenotype in the human immunodeficiency virus type-1 subtype C gp120. Virology, 409(2), 163-174.

3 Medical Dictionary. (n.d.). Retrieved October 18, 2016, from http://medical-dictionary.thefreedictionary.com/

4 Lackie, J. M. (Ed.). (2007). The dictionary of cell & molecular biology. Academic Press. http://site.ebrary.com/lib/loyolamarymount/reader.action?docID=10188656&ppg=4

5 Oxford Dictionaries - Dictionary, Thesaurus, & Grammar. (n.d.). Retrieved October 18, 2016, from https://en.oxforddictionaries.com/

6 1.2. Definitions: Pathogenicity vs virulence; incidence vs prevalence. (n.d.). Retrieved October 18, 2016, from http://www.coloss.org/beebook/II/virus/1/2

Useful Links

Courtney L. Merriam

Clas Page: Bioinformatics Laboratory

• Courtney L. Merriam
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