Christian Matranga
Sibylla Braedel
Johannes Ries
Since the early eighties, many different types of experimental HIV
vaccines have been proposed.
Among these are inactivated whole viruses, recombinant live viruses,
envelope proteins, etc These vaccines qualify for human use only in
cooperation with the Food and Drug Administration (FDA)(1).
If the vaccine passes FDA approval for human use, it is administered in three phases. Phase I and II focus on the dosage, safety, and immune response to the vaccine. A greater dosage sample is used in phase II than that of phase I. Phase III determines the relative ability of the vaccine to control infection (1).
Recently over 2000 volunteers in the U.S. have experimented one or more HIV type-1 vaccines at phase I and II levels. One of these volunteers, designated 2018BP was part of continued evaluation which spanned a three year period.
2018BP was a high risk participant for vaccination. The volunteer reported involvement in IV drug use and sexual intercourse with a HIV positive partner over a nine month period. A few days after the last sexual encounter, 2018BP complained of symptoms synonymous with HIV infection. By this point in time, the volunteer receiving phase II trials of recombinant HIV-1 mn rgp120 over a two year span(2).
A series of tests including HIV-1 RNA RT (reverse transcriptase) PCR amplification, an extensive test for viral RNA in serum and plasma, were performed immediately after suspected infection. Results from these test showed inconsistencies. For example, a sample of plasma from day 832 was HIV-1 positive by RNA RT PCR, yet one week later on day 838, the plasma read negative. Other tests such as western blot , ELISA, and DNA PCR were also negative for the appearance of HIV antibodies and serum viraemia(2).
These results draw many possible conclusions. 2018BP could be seronegative due to vaccine induced immune response and aborting of virus. This is reasonable to conclude because the behavior of 2018BP was so high risk. Yet the false positive reading (i.e. day 832) display difficulties in diagnosing HIV infection after vaccine administration.
To distinguish vaccination from infection, a new proposed vaccine is under inspection. The vaccine omits the gp 41 epitope found in all HIV positive serum. The gp 41 recombinant will provide specific and sensitive results in all assays(2).
Monoclonal Antibodies to HIV Type 1
Recently a study was conducted at the National Institute of Health (NIH) in Maryland on the effectiveness of monoclonal antibodies on HIV-1 virus isolates. The study was to determine which human monoclonal antibodies had the greater ability to neutralize an array of isolates. To be tested in humans, antibodies will display "90% neutralization of 70% of the primary isolates tested" at a low concentration (approximately 25 ug/ml)(3).
Seven monoclonal antibodies were provided, separated by HPLC for purification. Viral stocks included 10 isolates, nine clade adult and pediatric, and one laboratory isolate (MN). Neutralization assays were all PBMC (peripheral blood mononuclear cell) based.
Biochemical results determined by HPLC showed three of the seven antibodies eluted as single peaks, making up 100% of each peak area. OD readings at 280nm, BCA, and radial immunodiffusion were used to detect immunoglobulin concentration; two of the three pure monoclonal antibodies, IgG1b12 and 2G12 had consistent protein readings. Neutralization results showed 4 of the 9 viral isolates were neutralized at less than 25 ug/ml concentration of antibody. Some viral isolates were more susceptible to neutralization (3).
In conclusion, antibodies used in this study were human monoclonal antibodies to prevent development of host antibodies during possible clinical trials. No antibody met the criteria of neutralization (explained above), but IgG1b12 and 2G12 pure monoclonal antibodies neutralized greater than or equal to 4 of the 9 primary isolates at a low concentration. This changed the proposal for clinical trials to neutralization of 50% of primary isolates at 25 micrograms per milliliter or less. Cocktails of two or more of these antibodies which (for example) target different epitopes will be likely in trials. Yet the fact that studies were performed in vitro and antibodies may have different effects in vivo should also be considered during trial(3).
The HIV-1 protein gp120 as a B-cell superantigen
"B cell superantigens are substances that induce B cell response by
binding to part of the Ig molecule distinct from the antigen binding
site."
Usually most of the B cell antigens, like staphyloccoccal protein A,
staphyloccocal enterotoxin and HIV gp120, are binding to a part of the
heavy chain, inducing a humoral response of the B cell without recognition
of antigen.(5)
The protein gp120 is a surface protein on the HIV-1 virus, trimers and
tetramers of gp120 are noncovalent associated to the membrane of the virus
by the transmembrane domain of gp41. Containing an epitope with the CD4
binding site, gp120 is important for the viral entry in T cells.(6) The
different subtypes in the AIDS epidemic have been assigned by the gp120
sequence homology, and these subtypes or clades are showing differences
in infectiousness and spreading on the continents. (7)
In the beginning of an infection with HIV, B cell activation occurs with an increased level of Ig production, epecially the level of VH3-B cells. After the outbreak of AIDS then, a progressiv depletion of CD4 T cells and parallel a depletion of VH3-B cells, with degeneration of germinal centers in lymphatic tissue, takes place. In "HIV seropositive individuals" an increase in VH3-B cells, in contrast to the normal level of VH3-Immunoglobulins of approximately 50% of all B cells in uninfected persons, was seen, but these level was markedly reduced in AIDS patients.(7)
In vitro studies have been shown, that the gp120 protein binds to 3-6%
(5-25%) of an uninfected B-cell population via the immunoglobulin,
compared to 0.01% of all B-cells who are able to recognize a novel
antigen. (5,6,7) It was also possible to stimulate B cells to secrete
VH3-Ig with gp120 and conditions to
mimic T cell help (anti-CD40 and IL-4). (7)
The exact gp120 binding site on the VH3-Ig is unknown, there is nevertheless an evidence where the gp120 is not binding, because the Ig light chain and the Fc portion are not involved, and Fab fragments have gp120 binding activity. Also it was experimentally proved, that gp120 does not bind at the "normal" CDR epitope for antigens. In these tests an antibody against the hapten dansyl was used, and it was determined, that the binding specificities did not overlap.(7)
In the study, "Identification of the B cell superantigen-binding site of
HIV-1 gp120" (4), the protein sequence of gp120 was examined to detect the
exact binding site for the Igs.
First, in preceding experiments, it was ascertained , that these B-cell
superantigen activity does not depend on the glycosylation or the molecule
confirmation, because neither the unglycosylated nor the denaturated
protein have a reduced activity in comparison to the native protein.
Then recombinant gp120 proteins of different origins were compared and it
was recogniced, that the gp120 protein sequence is well conserved.
The test for the protein sequence of the binding site itself was made with
an ELISA, in which a panel of synthetic peptides, which are covering the
whole sequence of gp120, and monoclonal IgM and IgG were used. Only three
of these peptides showed a superantigen binding with the Immunoglobulins,
and a mixture of them had no additional positive effect to the
superantigen binding.
This result indicates, that the superantigen binding site is on the V4
variable domain and the C2 constant domain of gp120, because these are the
location sites of the three peptides.
To examine these regions, different mutant peptides were synthesized, and
it was recognizd, that an amino-acid substitution in Leu 395, Asn 397,
Ile 425 and Gln 427 leads to an impaired or abolished superantigen
binding.(4)
The evidence, that gp120 is really a superantigen for B cells, is mostly indirectly based on the observation of VH3 restriction and depletion of B cells in vivo during an HIV infection, and the binding of gp120 to Ig and activation of B cells in vitro. But the exact molecular interaction of gp120 with the B-cell receptor is still unknown.(6)
CD4-induced interaction of primary HIV-1 gp 120 glycoproteins with the
chemokine receptor CCR-5
Introduction:
the infection of the host-cell with HIVs requires 1 two step process:
Attachment and entry.
First, the glycoprotein gp 120 on the virus envelope binds to the CD4
molecule on the target cell. But how can the HIV-1 efficiently enter into
the host-cell? It has been shown that therefore the complex of gp 120 and
CD4 interatcs with the chemokine receptor CCR-5 also on the target-cell.
This interaction inhibits the binding of the natural CCR-5 substrates. The
gp 120 or the CD4 alone have a much lower affinity to CCR-5 than the
complex.
What exactly is responsible for the interaction with CCR-5?
To figure it out some experiments - mentioned later - were done. Therefore
the following proteins were used:
Materials:
loops:YU2DC1DV1/2DC5 or
JR-FL contains the whole sequence, BAL has a deletion in C1,
YU2DC1DV1/2/3DC5 have deletions in all regions indicated with "D".
binding site,CD4 binding,
17b that binds a discontinuous gp 120 epitope exposed better after
CG10 that recognizes an epitope present onlu on gp 120-CD4
complexes,
19b and loop2 that recognize the V# loop of JR-FL gp 120 and L71 that can decrease gp 120-CD4 interaction in some contexts.It has already been known that all gp 120 derivatives bind CD4 with Kd = 4-30 nM and F105 and 17b antibodies in the absence and presence of sCD4.
Experiments:
In the first experiment of several gp120 derivatives with MIP-1alpha was
studied in the absence and presence of CD4. For that purpose the ligand
was radiolabeled. In the case of JR-FL, BAL and YU2 C1 V1/2 C5, the
presents or sCD4 increased the inhibition of binding of the natural ligand
100 to 1,000 fold. In the same experiment with YU2 C1 V1/2/3 C5, which has
no V3 loop, the inhibition of MIP-1alpha binding was very much reduced in
both the absence and the presence of sCD4. This indicated, that the V3
loop is necessary for the inhibition. When this experiment was done with
L1.2 cells expressing CCR-1, no inhibition happened. This was a proof for
the specificity for the CCR5F-L1.2 receptor.
Then the effect on the other natural ligands of CCR-5 were tested. The
inhibition of binding (MIP)-1beta and RANTES took place in the same case
as this of (MIP)-1alpha. In a second experiment D1D2sCD4 was compared with
CD4. The result was that D1D2sCD4 inhibited (MIP)-1alpha and (MIP)-1beta
binding much more in the absence of gp120. This inhibition was also
specific for CCR-5.
The next experiment studied the effect of anti-gp120 monoclonal antibodies
on the inhibition of (MIP)-1alpha binding to CCR-5 by the JR-FL derivative
in the presence of sCD4.
17b, 19b and loop2 blocked inhibition whereas CG10 decreased it. But when
the antibody specific to gp120 domains interacting with gp41 was used, the
inhibition by the gp120-sCD4 complex was not blocked. L71, which can
decrease gp120-CD4 interaction affected also the inhibition.
This shows that CD4 is necessary for the interaction between gp120 and
CCR-5.
Another experiment laid open that the increased amount of natural ligands
or YU2 C1 V1/2 C5, but not YU2 C1 V1/2/3 C5, decreased the binding of
radiolabeled gp120 in the presence of CD4. This manifested the above
results.
Results:
the two step process, that means attachment and entry, make it possible
for the gp conserved elements that interact with CCr-5 to remain
undetected by antiviral antibodies for a long period. This "protection"
ends by binding to the CD4 just before the HIV-1 enters the target-cell.
The major site for DDR-5 interaction must be on the gp 120 because this
protein has a low affinity to CCR-5 if CD4 is not present. CCR-5 is a
co-receptor for diverse HIV-1 strains and for SIV. Therefore it seems to
be well-conserved.
The binding region on gp 120 can be localized in the following way: it
must be some-where else but C1, V1, V2 or C5, it can not be3 on the
epitopes specific for 17b and V3.
V3 is important for the binding, because deletion of the V3 loop disrupts
CCR-5 interaction.
Because D1D2sCD4 expose a CCR-5-interacitve region better than CD4, the
native CD4 sequence might have a higher affinity for CCR-5 in presence of
gp 120.
All together, the necessary regions on gp 120 and CD4 seem to be quite
distant from the target-cell membrane. For that reason, the gp 120 - CD4
complex must undergo a rearrangement during the entry to get the best
CCR-5 binding.
1)Kuby, Janis. Immunology. New York: Freeman, 1997
2)Schwartz, David H, et al. Extensive evaluation of a seronegative participant in an HIV-1 vaccine trial as
result of a false positive PCR The Lancet. 26 July 1997: 256-2593)D Souza, M. Patricia, et al. Evaluation of monoclonal antibodies to HIV
type 1 primary isolates by
neutralization assays Journal of Infectious Disease. May 1997: 1056-1061