HIV Vaccine

Doug Hosack Doug! at nih.gov
Wed Nov 4 14:28:23 EST 1998


Matthew B. Maison wrote in message ...
>
>I am a undergratuate in Introductory to Immunology and would like some
>help with a project I am doing.  We have to write a proposal for an HIV
>vaccine and how it works with the immune system.  They don't have to be
>right (of course), and they could have even been tried before.  Can anyone
>give me some ideas?  I was hoping there was something out there that is
>being done now (a 'hot idea' per say) someone would know about, or even
>be doing. Thank you in advance.
>
>---------------------
>Matthew B. Maison
>maison at u.arizona.edu
>

Most approaches today seek to maximize cell-mediated responses rather that
humoral responses.   There is even speculation that antibody responses to
HIV are counterproductive.  It seems that HIV is designed to generate an
antibody response against useless regions on gp120 in order to protect more
critical regions.  For this reason, I'm sure that you'll find plenty of
advise about protocols to generate anti-HIV CTL responses.

I will suggest a (far-fetched?) protocol to generate an effective IgA
response to prevent sexually-transmitted HIV infection.  In short, it
involves the creation of recombinant bacteria that express a protein similar
to a critical region on gp120.  In productive HIV infection, the immune
system does not generate antibodies to this region, probably because of an
antibody response to an adjacent "decoy" epitope.  This protocol is designed
to present the critical epitope without decoy epitope interference.

The basis of the approach is based upon the ability of a protein known as
cyanovirin-N to bind gp120 and prevent infection by all strains of HIV-1,
HIV-2, SIV and FIV tested thus far.  For background, please read
http://www.ama-assn.org/special/hiv/newsline/special/jamadb/cvn.htm

In brief, the protocol seeks to raise mouse antibodies against cyanovirin-N,
and isolate those antibodies that bind the same site of cyanovirin-N as
gp120.  The variable region of these mouse antibodies are now "molecular
copies" of the critical region of gp120.  The protocol next decribes how to
engineer bacteria to express this "molecular copy."  By seeding mucosal
linings with these recombinant bacteria, an IgA response against the
molecular copy would be raised.  These IgA's would hopefully cross-react
with gp120, and have a inhibitory effect similar to cyanovirin-N itself . .
.

Like all "dime-a-dozen" ideas, this protocol makes a lot of assumptions, and
would probably be a fruitless pursuit in the lab/clinic.  However, I hope
it's sufficent for your project.

First, immunize mice with cyanovirin-N and test for an anti-cyanovirin-N
antibody response.  Remove the spleen from successfully immunized mice and
isolate the mRNA.  Use RT-PCR with primers flanking the mouse Fab' gene to
amplify the sequence encoding the variable regions of the repertoire of
mouse antibodies.  Now insert these genes into a bacteriophage and infect
agar plates of bacteria.  ( there are kits on the market specifically for
the purpose of expressing antibodies in E. coli phage).  Screen viral
plaques with radiolabeled cyanovirin-N to isolate phage expressing
anti-cyanovirin-N.  Now grow each candidate phage in bacterial culture and
reisolate the DNA encoding the peptide of interest.  Sequence each isolated
DNA,  and sythesize the corresponding peptides.  Test each candidate peptide
in a competitive ELISA against cyanovirin-N binding to gp120.  Any peptide
that inhibits cyanovirin-N binding to gp120 is either binding the same site,
or an adjacent site.  (All the other peptides bind other surfaces of
cyanovirin-N)  Splice the gene for each inhibitory peptide into an
expression vector for your "seed bacteria."   Pick a bacterium capable of
invading the mucosal surfaces, like Salmonella.  Using an adjuvant like
Cholera toxin which promotes IgA switching, vaccinate test subjects with the
various recombinant bacteria and test for the ability of the subjects' IgA
to inhibit HIV infection in vitro.  (Or vaccinate cats and test for
protection against FIV . . .)

Some caveats to consider:
Cyanovirin-N might exert its effect by crosslinking two areas on gp120 that
need to move apart to expose gp41.  By inhibiting this conformational
change, cyanovirin-N probably inhibits virion fusion to the target cell.  If
your mouse antibody only mimics one of these regions, that plaque would be
selected, and the resultant Fab' fragment might inhibit cyanovirin-N from
binding gp120.  However, the final bacterium would only illicit an antibody
response against the one region.  Therefore, any IgA made would not
crosslink gp120 and would not inhibit infection.  This is the reason for the
last step of testing each IgA batch against infection in vitro.

Also, note that the constant regions of mouse antibodies are immunogenic in
humans.  If the kit you choose expresses a peptide that includes some
constant regions in addition to the Fab' fragment, you will need to humanize
the gene (replace the constant regions with the human version).  Otherwise
you will get a predominant response to the mouse constant region and not the
region of interest.

I imagine that you might have your work cut out for you in working out the
details.  You might want to do some background research on anti-idiotype
antibodies before you begin.

Good luck!





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