Shivum Desai Journal Week 8

Individual Journal Assignment Week 8

Purpose

The purpose of this weeks assignment is to study the Kirchher paper, formulate an outline of the paper, a list of unknown definitions, and a powerpoint presentation presenting the paper's findings.

Methods

1. The first step of this weeks assignment is to read the Kirchher paper.
2. Next, an outline was made of the research paper, including all important information pertaining to previous studies, results, and discussion.
3. As the paper was read, a list of ten unknown scientific terms was formulated as well.
4. The last step of this weeks project involved making a powerpoint presentation that described the research article as a whole. Focusing on the tables and figures in the research paper that contains the project's results.

Outline of Kirchher Paper

1) INTRODUCTION

a) THIS STUDY LOOKS AT MULTIP[LE ENV GENES IN AN ASSAY OF AUTOLOGOUS AND HETERLOGOUS NEAUTRALIZATION FROM CHRONICALLY INFECTED SUBJECTS. THE QUESTION IS TO SEE IF THERE ARE COMMON NEUTRALIZATION SIGNATURES THAT ASSOCIATE WITH CROSS REACTIVE NAB RESPONSES AMOUNG THE C SUBTYPE OF HIV SEQUENCES.

b) NEUTRALIZING ANTIBODIES

i) THEY ARE AN IMPORTANT ROLE IN NEUTRALIZING HIV AND SHOULD BE THE MAIN TARGET OF A VACCINE.

ii) NABS CAPABLE OF TARGETING SUBTYPE C VARIANTS OF HIV WOULD BE THE BEST BECAUSE SUBTYPE C MAKES UP 50% OF THE VIRUS.

iii) CROSS REACTIVE NABS HAVE NOT HAD SUCCESS.

c) EPITOPES AROUND THE CD4 BINDING SITE OF GP120 COMPRISE KEY TARGETS FOR NEUTRALIZING HIV-1 POSTIVIE SERA.

i) NADS THAT BIND TO THIS REGION ARE THE MOST EFFECTIVE

d) 19 SITES IN GP120 AND 14 SITES IN GP41 WERE FOUND TO BE ASSOCIATED WITH NEUTRALIZATION SUSCPETABILITY OF VIRUSES THAT WERE MULTI-SUBTYPE.

e) STUDYING A SINGLE ENV GENE FROM A SINGLE SUBJECT IN A STUDY IS NOT VERY ACCURATE BECAUSE ENV GENE IS HIGHKY VARIABLE AND THIS VARIABILITY CAN LEAD TO DIFFERENT FUNCTIONS OF A HIV-1 INFECTION DUE TO CHANGES IN ITS GLYCOPROTEINS. \

f) SINGLE GENOME AMPLIFICATION WILL BE USED INSTEAD OF PCR BECAUSSE IT WILL PROVIDE MORE ORIGINAL AND ACCURATE RESULTS FROM THE INFECTED INDIVIUAL.

i) BETTER, MORE ACCURATE CHARACTERIZATION OF VIRAL GENE FUNCTIONS.

2) RESULTS

a) GENETIC ANALYSIS OF FULL LENGTH ENV GENES

i) SAMPLES TAKEN FROM 50 INDIVUALS IN NDOLA ZAMBIA IN 2005.

(1) 474 COMPLETE ENV GENES WERE OBTAINES FROM 37 OF THE 50 SUBJECTS. THE REMAINING 13 COUNDLT BE OBTAINED BECAUSE OF LOW PLASMA VIRAL LOADS.

(a) AN AVERAGE OF 13 EN SGA WERE SEQEUENCED FROM EACH OF THE 37.

(b) MOST OF SUBJECTS HAD SUBTYPE C.

(i) OTHERS HAD: D, A/C RECOMBINANT, OR G/J RECMBOINANT

ii) 13 OF THE 37 SUBJECTS SHOWED IDENTICAL OR CLOSELY RELATED ENV SEQEUCNES WHICH IS SUGGESTIN GOF CLONAL EXPANSION OF MINORITY POPULATIONS IN THE HIV 1 INFECTED INDIVIUALS.

iii) 9 SUBJECTS HAD CLONALLY EXPANDED ENV VARIANT

iv) FOUR SUBJECTS HAD TWO INDEPENDENT CLONALLY EXPANDED POPULATIONS.

v) FOUR OTHER SUBJECTS HAD SEQUENCES CLOSELY RELATED TO BUT BEGINNING TO DIVERGE FORM THE CLONALLY EXPANDED VARIANTS.

b) FUNCTIONAL ANALYSIS OF THE ENV QUASISPECIS IN INFECTED INDIVIDUALS

i) ENV PSEUDO TYPED VIRUSES WERE GENERATED BY COTRANSFECTING 293T CELLS WITH PCR PRODUCTS AND AN ENV DEFECTIVE BACKBONE PLASMID.

(1) INFECITIVTY WAS MEASURED BY LUCIFERASE ACTIIVTY IN TZM-BL CELLS.

(a) 377 WERE FOUND TO BE FUNCTIONAL. 80%.

(2) 8 OF THE SUBJECTS HAD ALL THEIR ENV GENES FUNCTIONAL WHILE THERE WERE A FEW THAT ONLY HAD A FEW FUNCTIONAL ENV GENES.

ii) 6 INDIVUALS WERE INFECTED WITH TWO LINEAGES OF VIRUSES.

(1) THIS DATA SUGGESED THAT WHILE SOME RECOMBINANTS COULD HAVE BIOLOGICAL ADVANTAGES, OTHERS MIGHT BE LETHAL AND RESULTS IN NON FUNCTIONAL GENES.

iii) THESE RESULTS SHOWED THAT INFECTIVITY WAS NOT DETEMINED BY EXPRESSION LEVELS OR THE CLEAVAGE EFFICIENCY OF GP 160 IN TRANSFECTED CELLS.

(1) THERE COULD BE A THRESHOLD LEVEL THAT NEEDS TO BE REACHED FOR THE PRODUCTIN OF INFECTIOUS PSEUDOVIRUSES SINCE ONLY A SMALL NUMBER OF ENV SPIKES ARE PRESENT ON MATURE HIV-1 PARTICLES.

c) AUTOLOGOUS AND HETEROLOGOUS NEUTRALIZATION

i) AN ASSAY CONTAINING AUTOLOGOUS AND HETERLOGOUS PLASMA SAMPLES WAS GENETERED AGAINST MULTIPLE REPRESENTATIVE ENV-PSUEDOTYPED VIRUSES FROM EACH SUBJECT.

d) A MAJORITY OF VIRUS/PLASMA COMBINATIONS (75%) WERE POSITIVE WITH MORE THAN 1/3 AT A NEUTRALIZATION TIER OF >100.

e) NEUTRALIZATION SIGNATURE ANALYSIS

i) THE CONSESUS SEQEUCENCES FROM ALL VARIANTS WAS EACH USED TO CAPTURE THE MOST COMMON AMINO ACID IN EACH POSITION.

ii) FOUR SEQUENCES WERE UNIDENTIFIED

(1) THREE IN GP120 AND ONE IN GP41

iii) THESE SITES HAD AMINO ACIDS THAT ASSOCIATED WITH STRONG OR WEAK SUBTYPE C CROSS REACTIVE NEUTRALIZATION

(1) ONE IS BELIEVED TO HAVE BEEN A FALSE POSITIVE

iv) THREE OF THE FOUR WERE FOUND IN THE V4 REGION. WHICH IS THOUGHT TO CONTRIBUTE TO PATTERNS OF NEUTRALIZATION SUSCEPTABILITY IN SUBTYPE C VIRUS.

v) PATTERNS SHOWED THAT ENVS ASSOCIATED WITH MORE CORSS REACTIVE NABS TENDED TO HAVE MUTATED TOWARDS AN ASN (N) POSITION AND GLY (G)

vi) PLASMAS THAT POSSESSED STRONGER NEUTRALIZING ACTIIVYT AGAINST HETERLOGOUS VIRUSES MORE POTENTLY NEUTRALIZED THE CONTEMPORANEOUS AUTOLOGOUS VIRUS.

vii) SIGNATUERE PATTERNS IN THE V4 LOOP ARE ASSOCIATED WITH POTENT AND CROSS REACTIVE NEUTRALIZING ANTIBODIES AGAINST SUBTYPE C VIRUSES.

3) DISCUSSION

i) ANALYSIS OF THE ENV GENE IN QUASISPECIES IN EACH INDIVIDUAL SHOWED CLONAL EXPANSIONS

(1) CORRELATION BETWENE VIRAL LOADS AND THE INFECTVITY OF ENV PSEUDOVIRUSES AND AMINO ACID SEQUENCES IN PLASMA WITH HIGH LEVELS OF CROSS REACTIVE HIV 1 NEUTRALIZATION ACTIVITY

(a) THIS IDENTIICATION HAS IMPLICATIONS FOR THE DEVELOPMENT OF EFFECTIVE HIV 1 VACCINES.

(2) ANALYSIS OF THE INFECTIVITY OF 474 ENV PSUEDOVIRUSES FROM 37 INDIVUALS REVEALED THAT A HIGH PERCNTAGE (80%) OF THE ENV GENES WERE FUNCTIONAL.

(3) PSEUDOVIRUSES GENERATED VIA SGA PCR PRODUCTS REVEALED A HIGH PERCENTAGE OF THE ENV GENES WERE FUNCTIONAL (80%)

(a) SAME RESULTS FROM SGA PCR DONE ON SMALLER GROUPS

(b) BUT DIFFERENT FROM BULK PCR OF ENV GENE (10%)

(c) DIFFERENCES IN NUMBERS MOST LIKELY DUE TO DIFFERENCES IN METHODS.

(4) ENV PROTEINS IN THE TRANSFECTED CELLS SHOWED THAT THE VARIATINO OF INFECTIVITY AMOUNG ENV PSUDOVIRUSES IN EAHC INDICUAL WAS NOT CORRELATED WITH IN VITRO EXPRESSION LEVELS.

(5) NONFUCTIONALITY OF THE ENV GENES MAY BE CAUSED BY MUTATIONS.

(6) POSITIVE CORRELATION TREND WAS OBSERVED BETWEEN THE LEVELS OF INFECTIVITY OF EN PSEUDOVIRUSES AND VIRAL LOADS IN THE DONOR SBJECTS.

(a) SUGGEST THAT VIRUSES WITH MORE INFECTIOUS ENV GENES MAY LEAD TO A HIGH LEVEL OF VIRAL LOADS IN VIVO

(7) DIVEREGENCE OF IDENTICAL SEQUENCES THAT STARTED OFF THE SAME INDICATES THAT THERE IS CLONAL EXPANSION OF SOME VIRUSES IN THE INFECTED INDIVIUALS.

4) RELATED RESEARCH

(1) RELATED STUDY

(a) CLONAL EXPANSION VIRUSES AND OTHER DIVERGENT VIRUSES WERE FOUND IN AN INFECTED MOTHER A DAY AFTER DELIVERY, BUT ONLY VIRUSES IDENTICAL OR HIGLY SIMILAR TO THOSE CLONAL EXPANSION VIRUSES WERE DETECTED IN THE INFECTED INFACT AT BIRTH.

(i) RESULTS SUGGES THAT CLONAL EXPANSION VIRUSES MAY PLAY AN IMPORTNT ROLE IN VIRUS TRNASMISSION

(2) THE CURRENT STUDY DOES NOT HAVE ANSWERS FOR CLONAL EXANSPION IN VIRUSES.

(3) RELATED STUDY

(a) NEUTRALIZATION SIGNATURE WAS INVESTIGSTED IN SUBTYPE C OF A 5 SUBTYPE PANEL B AND 5 SUBTYPE C VIRUSES.

(b) NO PATTERNS WERE FOUND TO ENABLE TRACKING OF ANY SIGATURE PATTERNS.

(c) A CORRELATION BETWEEN THE V1-V4 LOOP LENGTH AND CROSS SUBTYPE NEUTRALIZING POTENTIAL WAS OBSEVRED.

(i) CURRENT STUDY HAD NO SUCH EVIDENCE

(4) PREVIOUS STUDIES

(a) SHOWED AN ALPHA HELIX UNDER STRONG SLECTION PRESSURE, A REGION ASSOCIATED WITH RESISTANCE TO AUTOLOGOUS NEUTRALIZATION OF SUBTYPE C VIRUSES.

(b) FOUND C3V4 REGION LIKELY RESPONIBLE FOR INDUCING AUTOLOGOUS AND HETEROLGOUS NABS.

(c) DATA SUGGEST THAT SIGATURE PATTERNS IN THE V4 LOOP ARE IMPORTANT TO CONSIDER FOR THE DESIGN OF A VACCINE THAT CAN INDUCE POTENT AND BROADLY REACTIVE NABS AT LEAST FOR SUBTYPE C, WHICH IS THE MOST PREVALENT HIV 1 SUBTYPE IN THE WORLD.

Unknown Definitions

1. Glycosylation: The controlled enzymatic modification of an organic molecule, especially a protein, by addition of a sugar molecule.
   • https://en.oxforddictionaries.com/definition/glycosylation
2. MAb:Monoclonal antibody.
   • https://en.oxforddictionaries.com/definition/mab
3. 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.
   • https://en.oxforddictionaries.com/definition/bootstrap
4. Autolgous: (of cells or tissues) obtained from the same individual.
   • https://en.oxforddictionaries.com/definition/autologous
5. Quasispecies: Originally (in models of evolutionary selection): a group of similar but non-identical DNA or RNA molecules, with a distribution centred on a hypothetically ideal sequence, resulting from a balance between mutation and selection pressure. In later use also: a population of viruses with genomic sequences of this nature.
   • https://en.oxforddictionaries.com/definition/quasispecies
6. Clade:A group of organisms believed to comprise all the evolutionary descendants of a common ancestor.
   • https://en.oxforddictionaries.com/definition/clade
7. Luciferase Assays:
   • https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/reporter-gene-assays/luciferase-assays.html
8. Antigenicity: the capacity to stimulate the production of antibodies or the capacity to react with an antibody.
   • http://medical-dictionary.thefreedictionary.com/Antigenicity
9. Supernatant: the liquid lying above a layer of precipitated insoluble material.
   • http://medical-dictionary.thefreedictionary.com/supernatant
10. Amphotropism: indicates that a pathogen like a virus or a bacterium has a wide host range and can infect more than one species or cell culture line.
    • http://encyclopedia.thefreedictionary.com/amphotropic

Data and Files

HIV-1 Presentation

• Format of this presentation is slightly skewed due to file format versus presentation.

Conclusion

I started this weeks experiment by first reading the research paper assigned to my group. Then next step was to analyze all of the figures and tables and from there an outline was formulated that broke down all the important parts of the experiment, especially the discussion and related studies. These sections were important because the discussion section examined the evidence gathered by this study and the related studies showed the accuracy and relationship of this study in comparison to others. Next, definitions of words that were unknown to me were researched for clarification. The last step of this experiment was collaborate with my partners and form a powerpoint presentation that delivered all the information in the research paper, specifically focusing on the figures and tables.

Useful Links

[[Media: |Journal Week 9 Presentation]]

Week 8 Kirchherr Reading ²

References

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.

Acknowledgments

I worked with Matt, Avery, and Courtney on this weeks presentation project. I would like to thank them for their help on this project. While I worked with other people on this assignment, this individual journal entry was completed by me and not copied from another source.