# AVM BIOL368 Week 8

## Purpose

To prepare for a journal club presentation next week. Within our groups we will individually create an outline for the assigned paper. Then, as a group, we will create a powerpoint to present our topic to the rest of the class.

## Methods and Results

1. What is the main result (message) presented in this paper?
• Signature patterns present on sequences within the V4 loop may be associated with neutralizing antibodies (Nabs) and play an important role in prevention agains the HIV-1 virus, specifically subtype C.
2. What is the importance or significance of this work?
• It may help lead us towards a vaccination against AIDS.
3. What were the limitations in previous studies that led them to perform this work?
• There is high variability in the env gene, many studies did not have enough samples of env genes from each of their subjects. Other studies also did not perform SGA, which can "more accurately reflect what is present in vivo".
4. What were the methods used in the study?
• This study used amplification of HIV-1 env genes, infection assays, neutralization assays, western (protein) blots, sequence analysis, SGA, PCR, and clustering analysis.
5. Briefly state the result shown in each of the figures and tables.
• Table 1: Summarizes information from HIV-1 infected subjects including gender, age, subtype, viral load, SGA #, and functional env gene # and percentages.
• Figure 1: Unrooted phylogenetic tree of all completed env gene sequences from each subject.
• Table 2: Shows relative amount of clonal expansion sequences in a selection of subjects. This table includes subject ID #, SGA, Lucifer's activity, Total viral population percentages and # of amino acid differences among the env sequences.
• Figure 2: Rooted phylogenetic trees of 13 of the subjects. The trees were chosen to demonstrate clonal expansion within the env gene. Black dot markers are provided on the trees where sequences are identical.
• Figure 3: Infectivity of 474 env pseudoviruses from 37 individuals which was determined from TZM-bl cells. Values were measured by luciferase activity. Black lines illustrate when luciferase values were 10 fold greater than the control used.
• Figure 4: Correlation between viral load and infectivity of Env pseudoviruses. p=0.05
• Figure 5: Western blot analysis of HIV-1 protein expression in transfected cells. Expression levels of Env proteins are expressed as Env (gp160+ gp120):P24 ratios. The infectivity of the each Env pseudovirus is shown as relative light unit (RLU).
• Figure 6: Rooted phylogenetic tree of all SVA env genes from 15 subjects combined. Black markers are used to demonstrate low diversity within samples.
• Figure 7: Clustering of viruses based on neutralization.
• Figure 8: Signature amino acid sequences found in the V4 region and p41 associated with high levels of neutralization activity.
• Figure 9: Crystal structure of ligated gp120 with signature amino acids. B clade template shown, but not substantially different from C clade
• Figure 10: Comparison of autologous neutralizing activity between high and low heterologous neutralization plasmas. Plasmas with high neutralization of heterologous virus tended toward high neutralization of autologous viruses.
1. How do the results of this study compare to the results of previous studies.
• Their results differ with some studies, but also show promising results in comparison to others. One study suggested a correlation between the loop length of V1-V4 and neutralization potential, but this study could not find similar evidence. A previous study done by Moore et al. suggested a region in C3V4 had the potential to induce Nabs, and this study had similar findings in that the regions discovered are near that region and may have stronger Nab responses. This study agrees with previous studies that that the V4 loop may play an important role in the HIV-1 virus and should be studied further in the hopes of developing a vaccine.

# Outline

Introduction:

• Nabs, also known as neutralizing antibodies should be targeted by an AIDS vaccine
• HIV-1 has nine different subtypes
• This study focuses on subtype C
• subtype C infections account for 50% of HIV-1 infections
• most prevalent in countries such as Southern Africa and India
• autologous and heterologous Nabs are being studied in HIV-1 positive serum samples
• studies have shown that epitopes in the CD4 hold significant roles such as neutralization of HIV-1 positive sera
• different studies show high variability within neutralization
• too much reliance on PCR methodology for Env cloning in past studies
• env is highly variable and the study of this gene may result in missing information about the neutralization of epitopes
• This paper studies multiple env genes obtained via SGA methodology to determine whether there is neutralization signatures associated with cross-reactive Nab responses within subtype C viruses
• The study also searches for a signature sequence present among all the env sequences that could possibly be associated with neutralizing activity

Methods:

• 37 HIV infected individuals participated
• 474 env genes obtained from said subjects
• pPCR and SGA performed on each subject
• Western Blots were preformed
• 60 env-pseudotype viruses taken from 15 subjects were used to perform neutralization assays

Results:

• All but three subjects were infected with subtype C
• Other subtypes observed: D, A/C recombinant, G/J recombinant
• 13 subjects had identical or closely related env sequences
• frequent clonal expansion
• Of the 474 env genes observed, 377 were functional
• Some results suggested that some recombinant genes may gain biological advantages while some may becomes nonfunctional or lethal
• A positive correlation was found between pseudovirus infectivity and viral load
• Four signature sites were observed on the env gene
• three in gp120 and one in gp41
• 3/4 found in the V4 region
• q-values suggest at least one of the sites is a false positive
• Data points to association between signature patterns in V4 loop and Nabs against subtype C

Discussion:

• Analysis of env gene showed:
• high variability
• frequent clonal expansion
• high levels of cross-reactive HIV-1 neutralization activity within signature sites in the V4 loop
• correlation between viral load and infectivity of env pseudoviruses
• data showing high functionality of a majority of env genes
• Final results suggest that clonal expansion viruses may have an important (still unknown) role in virus transmission

## Data and Files

### Definitions

1. Contemporaneous:"Existing at or occurring in the same period of time."
2. Sera:"plural form of serum."
3. Epitope: "The part of an antigen molecule to which an antibody attaches itself."
4. Luciferase: "Any of the group of enzymes that act on the oxidation of luciferin of bioluminescent organisms."
5. Glycosylation: "The controlled enzymatic modification of an organic molecule, especially a protein, by addition of a sugar molecule."
6. Quasispecies:"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."
7. Amplicon: "Any PCR amplification product."
8. Bootstrap value:"a method for estimating parameters by repeatedly drawing random samples, with replacement, from the collected observations."
9. Antigenicity:"the property of being able to induce a specific immune response or the degree to which a substance is able to stimulate an immune response."
10. mAbs: "Monoclonal antibody."

## Conclusion

This paper was outlined and each figure was described in detail for preparation of our week 9 Journal Club. Our paper showed evidence of HIV-1 neutralization at signature sites within the V4 loop. It is important to take into consideration that this paper focused on subtype C, with only two of the subjects studied belonging to a different subtype. Subtype C accounts for more than 50% of the HIV-1 virus worldwide. Four signature sites were identified, three of them being within the V4 region of gp120 and the other being on gp41. Clonal expansion viruses were also followed as they are thought to play an important role in virus transmission. These results suggest that Nabs may be used in the advancement of a vaccine for AIDS. Future research would need to focus on the V4 loop and its correlation with neutralization signatures.

## Acknowledgments

Avery Vernon-Moore 22:14, 18 October 2016 (EDT)

## References

#### Weekly Assignments:

Assignment 1
Assignment 2
Assignment 3
Assignment 4
Assignment 5
Assignment 6
Assignment 7
Assignment 8
Assignment 9
Assignment 10
Assignment 11
Assignment 12
Assignment 14
Assignment 15


#### Individual Journals:

"Week 1: Create Wiki Page"
Individual Journal 2
Individual Journal 3
Individual Journal 4
Individual Journal 5
Individual Journal 6
Individual Journal 7
Individual Journal 8
Individual Journal 9
Individual Journal 10
Individual Journal 11
Individual Journal 12
No Week 13 Assignment
Individual Journal 14
Individual Journal 15


#### Shared Journals:

Class Journal 1
Class Journal 2
Class Journal 3
Class Journal 4
Class Journal 5
Class Journal 6
Class Journal 7
Class Journal 8
Class Journal 9
Class Journal 10
Class Journal 11
Class Journal 12
Class Journal 14
Class Journal 15