Results of the Transgenic Mouse Pilot Experiment

Figure 1

Neutralization of the homologous 168P PI virus

Transgenic mice (hu CD4+, hu CCR5+, mouse CD4+) were immunized with
FC immunogen COS-env with U87-CD4-CCR5 ( squares; n=3 ) or with cell controls U87-CD4-CCR5 cells alone or cocultured with mock-transfected COS cells (circles;n=3 mice).
Unimmunized mice were also used ( triangles;n=2 mice ). Sera was tested for neutralization of 168P with U87-CD4 cells expressing either CXCR4 (black symbols) or CCR5 (white symbols)

 

Need help with graph interpretation of Foci ( % control ) ?

In the neutralization assay, cells were infected with a known amount of virus, cultures were reacted with HIV antibody and a second (enzyme-linked) antibody. Foci represent clusters of infected cells detected by antibody similar to plaques or other CPE. % Foci is relative to a control well with virus but no antiserum.

Therefore, the lower the Foci, the greater amount of mouse sera antibody was able to neutralize the 168P PI viruses.

Fig. 1: Interpretations:

a) the fusion-competent (FC) vaccine immunogen ( squares; n=3 )

This FC immunogen = what mixture of cells? COS-env cells with U87-CD4-CCR5 cells

graph interpretation The homologous 168P PI virus was neutralized by mouse sera antibodies that could lock onto fusion-competent gp120

b) U87-CD4-CCR5 cells (circles;n=3 mice)

graph interpretation No inhibition of activity

What does this suggest? the transgenic mice were in fact tolerant to hu CD4 and CCR5 and that other adventitous cellular reactivites did not interfere with the virus infectivity assay

c) U87-CD4-CCR5 cells cocultured with mock-transfected COS cells (circles;n=3 mice)

graph interpretation No inhibition of activity

What does this suggest? the transgenic mice were in fact tolerant to hu CD4 and CCR5 and that other adventitous cellular reactivites did not interfere with the virus infectivity assay

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Stop and think #1: What do you think would happen if the fusion-competent (FC) vaccine immunogen had U87-CD4-CXCR4 cells instead of the U87-CD4-CCR5 cells?
answer Neutralization of the 168P virus by FC serum was observed regardless of the coreceptor used in the U87-CD4 cell infection assay ( Fig. 1 )

Stop and think #2: What does this suggest about the neutralization with CXCR4, a coreceptor to which the animal had not been exposed?
answer That neutralization does not directly target the CCR5 component of the vaccine.

Stop and think #3: In reference to figure 1, why the increase in Foci with dilution?
answer The serum antibody became more and more dilute in the serum. The higher the Foci, the lesser amount of mouse serum antibody was able to neutralize the 168P PI viruses.

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