Identifiable biomarker and treatment development using HIV-1 long term non-progressor sera

LTNP sera shows high antibody binding to recombinant HIV-1 gp 160 protein

Sera from five LTNP subjects (LTNP-1, LTNP-2, LTNP-3, LTNP-4, and LTNP-5) and normal
serum (NMS) were tested against the entire peptide library of HIV-1 MN Env gp160 protein
using an ELISA assay. All subjects showed antibody binding to the gp41 portion of
the gp160 protein, and sera from LTNP-2 and LTNP-3 had the highest antibody end point
titers (Figure 1).

Figure 1. LTNP Sera Analysis. Sera from five LTNP subjects (LTNP-1, LTNP-2, LTNP-3, etc.) and
normal serum (NMS) were tested against HIV-1 gp160 protein using an ELISA assay. All
5 subjects had been infected with HIV-1 clade B for over 15 years without developing
into AIDS. All subjects showed considerable antibody binding at the endpoint titer,
with subjects LTNP-2 and LTNP-5 (patient 25) having the highest antibody end point
titers.

LTNP sera samples epitope mapping and titer detection

After testing the sera against the whole peptide library of HIV-1 MN Env protein,
we successfully mapped peptides with high binding to those sera. Seven epitopes were
successfully mapped. The sequences and the homology results of a blast search are
listed here: Epitope A: QARLLLSGIVQQQMNLLRAT, 100% identical for 100 out of 100 sequences;
Epitope B: GCSGKLICTTTVPWNASWSNKSL, range from 95.7-100%, 20 out of 100 are 100%,
and 75 out of 100 are 95.7%, however the core epitope KLIC is 100% identical; Epitope
C: LLELDKWASLWNWFDITNWLW, 100%, 100 out of 100 sequences, Epitope D: DITNWLWYIKI 100%,
100 out of 100 sequences; Epitope E: WYIKIFIMIVGGLVGLRIVF, range from 90-100%, 3 out
of 100 are 90%, 82 out of 100 are 95%, and 15 out of 100 are 100%); Epitope F: PEGIEEEGGERDRDTRGRLV,
range from 85-100%, 8 sequences are 85%, and 92 out of 100 are 100%, and Epitope G:
IWVDLRSLFLFSYH, range from 92.9-100%, 2 out of 100 are 92.9 and the rest of them are
100%. Some peptides demonstrated binding to sera only at low dilution, while some
maintained high binding ability even at higher dilutions.

The following are the peptides (designated by NIH AIDS Research Reference Reagent
Program in 2004) to which patient LTNP sera demonstrated high binding at dilution
of 1:500: 2015 (TKAKRRVVQREKRAAIGALF ) (2), 2018 (SVTLTVQARL) (4), 2019 (QARLLLSGIVQQQNNLLRAI)
(5), 2020 (QQQNNLLRAIEAQQHMLQLT) (2), 2022 (VWGIKQLQARVLAVERYLKD) (5), 2025 (GKLICTTTVPWNASWSNKSL
) (4), 2031 (LLELDKWASLWNWFDITNWL) (2), 2032 (DITNWLWYIKI) (2), 2033 (5), 2038 (PEGIEEEGGERDRDTSGRLV)
(4), 2039 (RDRDTSGRLVHGFLAIIWVD) (2), 2040 (IWVDLRSLFLFSYH) (4), 2042 (HRDLLLIAARIVELLGRRGW)
(3), 2043 (IVELLGRRGWEVLKYWWNLL) (3), and 2046 (SLLNATAIAVAEGTDR) (2). The following
peptides bound to LTNP sera at 1:2000 dilutions: 2019 (4), 2025 (3), 2031 (3), 2032
(2), 2033 (3), 2038 (2), 2040 (3), and 2047 (AVAEGTDRVIEVLQRAGRAI) (2).

When mapping the LTNP samples, containing antibodies that bound with high affinity
at low sera concentration, we found that there was high protein homology between them,
representative of sequence conservation. These results are presented in Figure 2 and Table 1.

Figure 2. Schematic of gp160 of HIV-1 MN and epitope mapping results. The results for epitope
A-G are shown in the figure accompanying the structure for the glycoprotein 160. The
location and degree of overlap are shown in the figure also. The complementary information
on this figure is provided in Table 1.

Table 1. Summary of found epitopes from the study from LTNP sera

LTNP sera mediated inhibition of MT-2 infection cells by H9MN

Viral infection was inhibited both by cell-free LTNP sera and sera from mice vaccinated
with CL3 chimeric peptide (containing an HIV neutralization epitope). The Figure 3A demonstrates MT2 cell syncytia formation after infection by cell-free H9MN virus.
Sera from patient LTNP-2, LTNP-3, LTNP-4, and patient 25 (LTNP-5), from which the
human monoclonal antibody CL3 was developed, markedly inhibited syncytia formation
(p??0.05). In Figure 3B (right graph), this shows inhibition of viral infection using antisera generated
from vaccinated mice (AC peptide) binding to gp41 (Figure 3). All LTNP sera samples inhibited syncytia formation in vitro (Figure 3A). Due to the consequences, it gives credit to the idea that there is motivating
interest in studying LTNP sera and the potential for finding additional neutralizing
antibodies.

Figure 3. Inhibition of viral infection by LTNP sera and vaccinated mice sera. Viral infection
was measured after treatment with LTNP sera and sera from mice vaccinated with a peptide
containing an identified HIV neutralization epitope. A (left graph) demonstrated the MT2 cell syncytia formation after infection by cell
free H9MN virus. As shown in the figure, sera from patient LTNP-2, LTNP-3, LTNP-4,
and LTNP-5 (patient 25) significantly inhibited the syncytia formation (p??0.05).
B (right graph) shows antisera from the vaccinated mice (with the AC peptide) binding
to gp41, and comparing this binding titer to the positive control sera of patient
25 and the negative control sera of mice vaccinated with the beta amyloid peptide.

CL3 neutralized HIV-1 Clade B Clinical Isolate

The preparation of the monoclonal antibody has been described by Dr. Cotropia et al.
34]. The peptide GCSGKLICTTVPWNASWSNKSL, which has previously been determined to contain
the HIV-neutralizing epitope KLIC of the clone 3 (CL3) antibody, was used to vaccinate
mice in this experiment, and the antisera obtained from the mice were compared to
the titer of the CL3 antibody. Sera from patient 25 (LTNP-5), the sera from which
the human monoclonal antibody CL3 was developed, was used as a positive control. Sera
from mice vaccinated with the beta-amyloid peptide served as a negative control. We
generated several human monoclonal antibodies using sera from human patient 25 in
our previous study, Cao et. al. (2004) 35], and we found that CL3 binds to peptide 2025. This antibody can successfully neutralize
HIV-1 clade B clinical isolate (Figure 4). The neutralization profile of CL3 is provided, along with results for two other
established HIV-neutralizing HuMabs (2F5 and 2G12). The concentration at which CL3
inhibits 50% of cellular infections by the 92 BR030 isolate was 2 ?g/ml, while the
same value for 2F5 was 10 ?g/ml. This indicates that for this HIV-1 isolate, CL3 is
more effective than 2 F5 at neutralizing the infection.

Figure 4. Neutralization Profiles. The neutralization profile from CL3 is provided along with
results for two other established HIV neutralizing HuMabs (2F5 and 2G12). A CL3 and 2F5 are compared against the negative control, a control human monocolonal
antibody. B CL3 and 2F5 are compared against the positive control, 2G12. The concentration of
CL3 that inhibits 50% of cellular infections by the 92 BR030 isolate was 2 ?g/ml,
while the same value for 2F5 was 10 ?g/ml. This indicates that, at least for this
HIV-1 isolate, CL3 is more effective than 2F5 at neutralizing infection (p 0.05).

CL3 and 2F5 are conserved epitopes, implying their functional importance

Our results show that the CL3 and 2F5 epitopes are highly conserved among different
HIV-1 isolates, spanning clade A through U (Figure 5). The graph indicates the percent epitope homology among different HIV isolates across
clades A through U for HuMab anti-HIV neutralizing HuMab CL3 (epitope?=?8 aa long)
and 2F5 (epitope?=?6 aa long). The graphs indicate that CL3 consistently showed higher
conservation than 2F5.

Figure 5. Conservation of Epitopes across HIV-1 Clades A-U. As shown above, both the CL3 and
2F5 epitopes are conserved across all HIV-1 Clades A-U. CL3 appears to have a greater
percent homology across clades than the already established 2F5.

LTNP sera show few cell mediated (cytokine) differences from NHS

We conducted experiments regarding the levels of specific markers of the immune system,
mainly cytokines and chemokines. Many cytokines were measured using the RayBioTM Human
Cytokine Array Map V 5.1 (Figure 6). The data from the cytokine and chemokine array showed that chemokine CCL1 (I-309)
and insulin-like growth factor-1 (IGFBP1) showed significant decreases, while TIMP
metallopeptidase inhibitor 2 (TIMP-2) and neutrophil-activating protein-2 (NAP-2)
showed substantial increases (p??0.05) (Figure 6, Table 2). These results suggest that both the humoral and cellular responses in the immune
system of LTNP patients, mainly the LNTP sera (e.g. antibodies), may play a more significant
role in preventing disease progression.

Figure 6. RayBio^TM Assay Human Cytokine Array. Select result of proteins that showed a change in expression
can be found in Table 2 and manufacturers map of the array.

Table 2. Protein changes in LTNPs samples versus normal control