Last week researchers from Duke University reported that several
antibodies targeting phospholipids can potently inhibit HIV replication by an
indirect mechanism. Phospholipids are components of cell membranes that under
certain circumstances can be targeted by antibodies; in some cases this
phenomenon is associated with autoimmune disease (such as lupus) but
anti-phospholipid antibodies have also been found in people with no health
problems.
The Duke researchers identified four antibodies that showed very
strong anti-HIV activity in some, but not all, in vitro tests. Three of the
antibodies had been isolated from individuals with autoimmune disease while one
came from a healthy subject. Further analysis to ascertain why the antibodies were
only working in some tests revealed that the anti-HIV activity was indirect;
the antibodies worked by stimulating monocytes to produce beta-chemokines
(mainly two beta-chemokines called MIP-1 alpha and MIP-1 beta) and these chemokines
prevented CCR5-using HIV isolates from gaining entry into target cells (the
antibodies had no effect on X4-using HIV isolates). Because of this indirect
mechanism, the antibodies only showed activity if the in vitro tests included
monocytes. One standard test for measuring neutralizing antibody activity,
called the TZM-bl assay, does not include monocytes and therefore the
anti-phospholipid antibodies showed no activity when assessed by this method.
The researchers suggest that this novel mechanism of HIV
inhibition may be able to contribute to the development of vaccines and new
therapies but also acknowledge that: “the ability of such antibodies to stimulate
similar effects in vivo has yet to be determined.” It is unclear if the
induction of similar antibodies by vaccination would be expected to raise
systemic levels of beta-chemokines or would somehow induce their production
only upon HIV exposure (or some combination of these two possibilities).
Interestingly, there have also been reports recently that
anti-phospholipid antibodies can directly inhibit HIV. The postulated mechanism
for this activity is the binding of antibodies to cellular phospholipids that
HIV incorporates into its viral membrane when budding out of infected cells. At
CROI in San Francisco earlier this year, Heidi Reil showed that some antibodies
against the E2 protein of a virus called GBV-C bind phospholipids and can also
inhibit HIV (in the TZM-bl assay). Reil’s talk is available among the CROI webcasts (the last presentation in the Thursday abstract session on
HIV vaccines). Subsequently, at the Keystone vaccine conference a few weeks ago,
a US-based research group reported data independently confirming Reil’s
findings (see abstracts appended below).
J Exp Med. 2010 Apr 7. [Epub ahead of print]
Moody MA, Liao HX, Alam SM, Scearce RM, Plonk MK, Kozink DM,
Drinker MS, Zhang R, Xia SM, Sutherland LL, Tomaras GD, Giles IP, Kappes JC, Ochsenbauer-Jambor
C, Edmonds TG, Soares M, Barbero G, Forthal DN, Landucci G, Chang C, King SW,
Kavlie A, Denny TN, Hwang KK, Chen PP, Thorpe PE, Montefiori DC, Haynes BF.
Duke Human Vaccine Institute and 2 Department of Pediatrics,
3 Department of Medicine, 4 Department of Pathology.
Traditional antibody-mediated neutralization of HIV-1
infection is thought to result from the binding of antibodies to virions, thus
preventing virus entry. However, antibodies that broadly neutralize HIV-1 are
rare and are not induced by current vaccines. We report that four human
anti-phospholipid monoclonal antibodies (mAbs) (PGN632, P1, IS4, and CL1)
inhibit HIV-1 CCR5-tropic (R5) primary isolate infection of peripheral blood
mononuclear cells (PBMCs) with 80% inhibitory concentrations of <0.02 to
approximately 10 microg/ml. Anti-phospholipid mAbs inhibited PBMC HIV-1
infection in vitro by mechanisms involving binding to monocytes and triggering
the release of MIP-1alpha and MIP-1beta. The release of these beta-chemokines
explains both the specificity for R5 HIV-1 and the activity of these mAbs in
PBMC cultures containing both primary lymphocytes and monocytes.
Keystone Symposia: HIV Vaccines (X5), March 21-26, 2010, Banff,
Alberta, Canada
POSTER NUMBER: 309
GB Virus C envelope protein E2 elicits antibodies that react
with a conserved antigen on HIV-1 particles and that broadly neutralize HIV-1
infectivity
Emma L. Mohr, Jinhua Xiang, James H. McLinden, Thomas M.
Kaufman,
Qing Chang, Donna Klinzman, Jack T. Stapleton
Objective: The development of HIV-1 vaccine antigens that
elicit neutralizing antibodies to diverse HIV isolates has proven
difficult. Prolonged survival in
HIV-infected individuals is associated with the presence of antibody to a
common human flavivirus (GB virus C), and the GBV-C envelope glycoprotein (E2)
inhibits HIV-1 entry in vitro, raising the possibility that there is molecular
mimicry between GBV-C E2 and HIV-1 particles.
Methods: We
examined the HIV-1 neutralizing capability of GBV-C E2 antibodies elicited
during natural infection, murine monoclonal GBV-C E2 antibodies generated by
DNA immunization, and rabbit polyclonal GBV-C E2 antibodies generated by
recombinant E2 immunization. This
neutralizing activity and ability to immunoprecipitate particles by the panel
of antibodies was characterized for diverse multiple HIV-1 isolates and other
viruses, and pseudoparticles.
Results: GBV-C
E2 antibodies inhibited the replication of diverse HIV-1 isolates (3 clades,
and laboratory and clinical strains) in vitro, but not Yellow Fever Virus or
Mumps virus. HIV-enveloped HIV
pseudoparticles but not HIV-enveloped VSV pseudoparticles were also inhibited
by GBV-C E2 antibodies, suggesting that neutralization is not HIV envelope
dependent. E2 antibodies
precipitate HIV-1 particles including defective HIV particles with no envelope
glycoprotein, or with VSV, HIV or GBV-C envelope displayed on the
particle. Incubation of the
antibody with the virus particles prior to infection and maintenance of the
antibody in culture media suggest that the antibody inhibits entry of the virus.
Conclusions:
Together, these data suggest that GBV-C E2 antibodies neutralize a
diverse panel of HIV-1 isolates by interacting with a conserved, non-gp160
antigen on HIV-1 particles. This
antigen appears to be exposed during virus particle budding, because
permeabilization of cells increases cross-reactivity of these antibodies with
cells. These data indicate that
GBV-C E2 protein, a non-HIV-1 immunogen, induces antibodies that cross-react
with and neutralize the HIV-1 infectivity, and suggests that these antibodies
may be causally related to the delayed mortality observed in clinical
studies. Immunization studies in
rhesus macaques is underway.
Keystone Symposia: HIV Vaccines (X5), March 21-26, 2010, Banff,
Alberta, Canada
POSTER NUMBER: 415
GB Virus C envelope protein E2 elicits polyvalent antibodies
that cross-react with HIV-1 gp41 MPER (T-20) and lipids
James H McLinden, Jinhua Xiang, Emma L Mohr, Thomas M
Kaufman, Qing Chang, Jack T
Stapleton
Objective: GB
virus C (GBV-C) is associated with prolonged survival in HIV-infected
people. GBV-C envelope
glycoprotein E2 inhibits HIV-1 entry, and antibodies to E2 protein neutralize
diverse HIV-1 isolates in vitro.
Broadly neutralizing anti-HIV human monoclonal antibodies (MAbs) have
been identified that bind to the MPER region of HIV-1 gp41, which includes the
HIV fusion inhibitor peptide sequence (T-20). Thus, E2 may share structural similarities with HIV proteins
Methods:
Previously described murine MAbs to E2 (M1VS, M6, M11), rabbit
antibodies (Abs) generated against recombinant E2 (rE2) or a synthetic peptide
recognized by an anti-E2 murine MAb (rPep), and human anti-HIV MAbs (2F5, 4E10,
2G12) were tested for reactivity against E2, T-20 (NIH repository catalog
#9845) and phosphatidyl-inositols (PI) with varying phosphorylation by either
solid phase or MAb capture ELISA.
Relative binding affinity was estimated by determining the Ab
concentration required for half-maximal absorbance in a given assay, and this
was not adjusted for the polyclonal rabbit IgGs.
Results: GBV-C
E2 antibodies all bound E2 as expected.
Surprisingly, the HIV MAbs 2F5 and 4E10 also bound GBV-C E2, in some
cases with more affinity than the E2 antibodies (relative E2 binding of
antibodies:
rE2>2F5>M11>rPep>M1>4E10>M6). HIV MAbs 2F5 and 4E10 bound T-20 as
expected, and 3 of the E2 antibodies also bound T-20 (M6, rPep, and rE2), although
the relative affinity of these 3 antibodies was > 100-fold less than the two
HIV MAbs. The anti-GP120 MAb 2G12
did not bind T-20 or E2. 4E10 and
M6 bound nonphosphorylated PI and PI with 3 or 1 phosphates respectively. E2 extracted with chloroform to remove
lipids led to a reduction in antibody binding by 58-fold.
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