ACS Medicinal Chemistry Letters
Letter
to include a recombinant envelope glycoprotein E2 antigen
control for all neutralization experiments following literature
protocols. Positive neutralization, defined as >50%, was
2
3
30
without the transmembrane domain, namely E2ΔTM3 . For
the purposes of this study, we elected to use a single antigen
E2ΔTM3, which has previously been effective in producing
broadly neutralizing antibodies against HCV1 (Genotype 1a
highest for the purified polyclonal Ab pooled serum sample of
3 with 78% neutralization at a concentration of 1 μg/mL. This
was statistically significant relative to the tucaresol control at
the same concentration. For each of the samples tested, high
end point titers also correlated with high % neutralization data
23−27
strain) in both rodents and humans.
The E2ΔTM3
protein comprises the complete ectodomain; it also ensures
that all the antigenic sites are accessible on the E2 core and
that the protein can be readily taken up by cells. This
technique has been widely applied to many E2 con-
(
P < 0.0016) (Table 2).
Table 2. Left: In Vitro Pseudotype Virus Particle
1
4,28,29
structs.
A well-established tucaresol mouse vaccination schedule
a
Neutralization with Pooled Immune Sera. Right:
Correlation of Individual (End Point Titer Values EPT)
with Neutralization % Values for Each Mouse
17
initially pioneered by Rhodes was used in our HCV testing.
Thus, each priming HCV vaccine was formulated with 25 μg of
E2ΔTM3 antigen and 200 μg of experimental tucaresol
adjuvant administered on day one. Mice then received 200 μg
doses of adjuvant only on days 2−5 thereafter to give 1 mg of
adjuvant per mouse per vaccination period evenly split over
five consecutive days. Booster injections were performed under
the same schedule at 4-week intervals post prime injections,
except with 5 μg of E2ΔTM3 antigen given on the first day.
BALB/C mice at weeks 0, 4, 8, and 12 (see SI). Adjuvant
positive controls included tucaresol, and the negative control
included E2ΔTM3 without adjuvant. Bleeds were performed
before the priming injections and 2 days after the booster
injections. Mouse weight was monitored on a weekly basis, and
infection at the site of injection was noted (SI)
a
A one-way ANOVA was performed for each immunization group,
followed by a Dunnett’s post hoc comparison test, respectively. *P <
0
were averaged for each group (n = 4).
.05. Significance is denoted by an asterisk (*). Neutralization values
Taken together, these data suggest that introducing a
simplified lipid-adjuvant based system positively affects
autologous neutralization and presents itself as an alternative
approach for rational HCV vaccine design, potentially
overcoming the need for elaborate vaccine delivery systems
and complex adjuvants. From a tucaresol scaffolding stance,
the aldehyde appears to be essential for a robust immune
response. Moreover, regiochemistry of the phenol in relation-
ship to the aldehyde was also critical with the para position
most favorable, followed by ortho relative to the aldehyde.
For this structural class, the change in physiochemical
Antibody end point titers in response to vaccination were
elucidated by an ELISA-based measurement of sera IgG
specific to the native E1E2 antigen. Adjuvant 3 (Table 1)
Table 1. Average Serum IgG End Point Titers (Left) and
Error Measurement (Right) Calculated at 3× Background
Absorbance for Individual Mice (n = 4) for Each Group
End point titer values
Adjuvant
Tucaresol
Bleed 2
Bleed 3
4471 ± 1489
17295 ± 10253
18777 ± 8992
5740 ± 5470
1883 ± 1578
3967 ± 1127
8021 ± 3288
17623 ± 9902
8404 ± 2286
5862 ± 2464
185 ± 50
74493 ± 39166
253271 ± 189742
290929 ± 149783
8900 ± 7851
properties by alteration of pK (increase in acidity) as seen for
a
1
3
6
8
1
1
2
1
1
2
ortho hydroxyl analogues 12, 18, 19, and 22 may be
responsible for the toxic effects. In addition, the possibility of
intramolecular H-bond formation by interaction of the ortho
phenol with the aldehyde carbonyl may present additional
effects such as assistance with Schiff-base formation, catalytic
effects, and subsequent increase in lipophilicity. Strengthening
this conclusion, meta substitution of a phenol as seen for 6 was
detrimental toward the immune response and no toxicity was
observed. Although toxic effects were not observed for both
the benzoic (16) and acetophenone (11) structures; with these
derivatives a downward trend was seen with the immune
response, further strengthening the argument of Schiff-base
formation. Moreover, steric effects, i.e. positioning of the lipid
chain, are best seen by comparison of adjuvants 3 and 8. Here
a significant difference in immune response is observed for 3,
but both showed no toxicity despite the ortho phenol
displayed in 8. Notably, the end point titer values were 3-
fold greater relative to parent tucaresol, which demonstrates
that 3 is the best adjuvant in this structural class.
3051 ± 2202
1
6
2
9
8
15869 ± 4507
32083 ± 13151
281958 ± 158437
33616 ± 9145
23449 ± 9854
21 ± 8
E2ΔTM3
generated the highest E2-specific serum IgG titers, thus
appearing to be the superior adjuvant based upon this
ELISA data. Compound 3 also produced the least injection
site infection. While adjuvants 22 and 12 revealed highly
comparable end point IgG values to adjuvant 3, infection at the
site of injection was markedly greater.
Based upon ELISA data, the best performing adjuvants were
then tested for their ability to neutralize HCV pseudotype virus
infectivity in vitro. E1E2 glycoproteins were expressed from an
autologous genotype 1a strain HCV (H77) and an unrelated
envelope glycoprotein from lymphocytic choriomeningitis
virus (LCMV), the latter being used as a negative control
In summary, we have developed a new adjuvant platform
and designed a vaccine formulation for neutralizing Ab
responses against HCV in vivo. In particular, adjuvants
containing E2ΔTM3 antigen elicited promising E2-specific
IgG titers with superior neutralizing capacities compared to
(SI). Monoclonal antibody (mAb)19B3 was used as a positive
C
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
Belz, Tyson F.
