Matthew R Allegretti Week 8

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Week 8 Assignment

Biological Terms

  1. mAbs: Monoclonal Antibody-A substance, usually a protein, which can be synthsised in the laboratory in pure form by a single clone (population) of cells. These antibodies can be made in large quantities and have a specific affinity for certain target molecules called antigens which can be found on the surface of cells and those that are malignant. Monoclonal antibodies are currently being investigated as a possible form of cancer treatment although their benefit has not be fully proven.
  2. Cross-reactive: antibodies which don't respond to any one specific antigen, but will respond to a number of them. These antibodies can be responsible for false positive results in antigen-antibody tests.
  3. Recombinant: A cell or an individual with a new combination of genes not found together in either parent, usually applied to linked genes.
  4. Clonal expansion: the multiplication of lymphocytes after their activation by antigen, so that large clones of rare antigen specific lymphocytes are generated to fight the infecting pathogen. (Parham 2014).
  5. Amplicon: a term for any small, replicating dna fragment.
  6. Autologous: Derived from an organisms tissues or dna.
  7. Heterologous: Of, or relating to, tissues or cytologic elements not normally found parts of the body of an individual, or that are derived from a different species.
  8. Titer: an emperical measure of avidity of an antibody.
  9. Transfection: The process of deliberate introduction of nucleic acids into a recipient eukaryotic cell.
  10. Luciferase: Any of the group of enzymes that act on the oxidation of luciferin of bioluminescent organisms.


Identification of amino acid substitutions associated with neutralization phenotype in the human immunodeficiency virus type-1 subtype C gp120


  • Study is significant because a cross-reactive neutralizing antibodies may be a possible method of vaccinating HIV-1
    • HIV-1 is quite diverse, but if a vaccine could effectively target even 1 of the 9 clades would be substantial in areas where that clade dominates.
  • Nabs targeting C subtype would be most desired
    • C subtype approximately 50% of infections worldwide
      • Dominates large regions in South Africa and India
  • Initial efforts to create cross-reactive Nabs had limited success
  • Studies point to the viablility of this approach
  • Epitopes around gp120 binding sites are key targets
    • Other important areas still need to be identified
    • Multiple regions are targeted
  • 19 regions in gp120 vulnerable to neutralization activity, 14 in gp41
    • Previous studies focused on limited numbers of env genes, which are highly variable, so information about neutralization epitopes is limited.
  • Previous studies were also limited by bulk PCR methodology
    • Improve upon the methods by using SGA (single-genome amplification)
  • Sequenced env genes from 37 infected individuals and characterized by infectivity and susceptibility by neutralization.
  • V4 region of gp120 associated with cross-reactive Nab responses in subtype C HIV-1-infected individuals
    • Shown to be important for Nab susceptibility in subtype C


  • Plasma samples collected from 50 chronically infected individuals
    • 474 complete env genes collected from 37 of the 50 subjects
      • Some did not return sequences because of low or undetectable viral loads
    • Majority of subjects infected with HIV subtype C (34 of 37)
  • Findings suggest frequent clonal expansion of minority viral populations
    • These minority clones recombine with other viruses to generate significant increases in diversity
  • Infectivity measured by luciferase activity in TZM-bl cells
  • Recombinant genes occur
    • Some confer biological advantages
    • Others result in non-functional genes, or lethal recombinations
      • Recombination of clonal expansion viruses with other viruses resulted in a wide range of infectivity
  • It was difficult to determine whether infectivity was associated with protein expression levels or plasma viral RNA load
  • Results suggest that variation in infectivity was not influenced by expression levels or cleavage efficiency of gp160 in transfected cells
  • Plasma samples were separated into low and high neutralization potency groups (fig 7)
  • 4 signature sites ID'd, 3 in gp120, one in gp41
    • At least one identified as a false positive.
  • V4 region, in association with the C3 alpha-2 helical domain important to virus susceptibility in subtype C
  • Autologous neutralizing activity significantly higher in high neutralization plasma group than low neutralization groups
    • "plasmas that possessed stronger neutralizing activity against heterologous viruses more potently neutralized the contemporaneous autologous virus"
  • Data suggests patterns in V4 loop is associated with cross-reactive Nabs against subtype C


  • Infectivity of env gene not correlated with in vitro expression levels
  • Clonal expansion is occurring in some individuals
    • Supported by multiple quasispecies with near-identical sequences
    • Clonal expansion may be integral to virus transmission
      • Another study will be needed to test this
  • Signature of four amino acids associated with potent neutralizing plasmas found
    • three of four were near the base of the V4 loop
    • Glycine at several positions in the V4 loop could lead to conformational elements and neutralization of antibody
  • potential glycosylation site 413 is, in fact, glycosylated
    • Not clear how this relates to neutralizing antibodies
    • Position is quite variable
  • correlation between the V1–V4 loop length and the cross-subtype neutralizing potential was observed in previous studies
    • Not supported by this study's findings
      • May be due to underpowered number of samples
      • Alternatively may be caused by the chronic nature of HIV infection in subjects
  • Previous studies show alpha-2 helix is under strong selective pressure
    • Important, as region may be related to autologous neutralization of C subtype
    • V4 region is downstream from the alpha-2 helix
      • Structurally related
  • The newly identified three signature amino acids were located in the vicinity of the previously reported region targeted by Nabs
    • signatures may be responsible for eliciting broader and more potent Nab responses or represent the escape mutations from the broadly Nabs against subtype C viruses through similar pathways
      • signature patterns in the V4 loop are important to consider for the design of a vaccine that can induce potent and broadly reactive Nabs

Methods and Materials

  • Plasma samples collected from 50 chronically HIV infected individuals in Ndola, Zambia in 2005.
    • Already enrolled in another HIV study
    • Approved by numerous ethics committees
    • None were treated with anti-retroviral drugs
    • Most likely infected by heterosexual transmission
    • Multiple rev/env genes from each individual were obtained by using single-genome amplification
  • pPCR products were cotransfected with an env-deficient HIV-1 backbone plasmid
    • With the addition of a number of other reagents, luminescence was eventually measured
    • 48 hours after cotransfection, cells were lysed and fluorescence detected, and density of env genes determined
  • Sequences of SGA env amplicons were obtained by cyclesequencing and dye terminator methods
  • Hierarchical clustering analysis was performed
  • Signature analysis was performed
    • envelope sequences were divided into two groups
    • Sequences were aligned and phylogenetically corrected
  • Nucleotide accession numbers available at genbank

Figures and Tables

  • Table 1: Summarizes information about all the subjects including gender, age, subtype, etc.
  • Figure 1: Phylogenetic tree of env gene sequences from all subjects
  • Table 2: Shows relative amount of clonal expansion sequences in various subjects.
  • Figure 2: Phylogenetic trees of 13 of the subjects- focused on clonal expansion genes
  • Figure 3: Infectivity of 474 pseudoviruses from 37 individuals, measured by luciferase activity
  • 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: Phylogenetic tree of all env genes from 15 subjects.
  • Figure 7: Clustering of viruses based on ability to neutralize
  • Figure 8: Signature amino acid sequences associated with high levels of neutralization activity. Key positions are highlighted
  • 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


Week 8 Presentation


While I worked with the people noted above, this individual journal entry was completed by me and not copied from another source. We met once outside of class to prepare our presentation and communicated electronically.

  • Matthew R Allegretti 23:52, 24 October 2016 (EDT)
  • Dr. Dahlquist: I pulled my reference formatting for the article from the week 8 page


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