JVI Accepted Manuscript Posted Online 11 April 2018 J. Virol. doi:10.1128/JVI.00369-18 Copyright © 2018 Bricault et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
1
Neutralizing Antibody Responses following Long-Term Vaccination with HIV-1 Env gp140 in
2
Guinea Pigs
3 Christine A. Bricaulta, James M. Kovacsb,c, Alexander Badamchi-Zadeha, Krisha McKeed,
5
Jennifer L. Shieldsa, Bronwyn M. Gunne, George H. Neubauera, Fadi Ghantousa, Julia Jenningsa,
6
Lindsey Gillisa, James Perrya, Joseph P. Nkololaa, Galit Altere, Bing Chenc, Kathryn E.
7
Stephensona,e, Nicole Doria-Rosed, John R. Mascolad, Michael S. Seamana, Dan H. Baroucha,e#
8 9
a
Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Boston, MA,
10
02215, USA
11
b
Department of Chemistry and Biochemistry, University of Colorado, Colorado Springs, CO,
12 13
80918, USA c
Division of Molecular Medicine, Children’s Hospital, Boston, MA 02115, USA; Department of
14 15
Pediatrics, Harvard Medical School, Boston, MA 02115, USA d
Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National
16
Institutes of Health, Bethesda, MD, 20892, USA
17
e
Ragon Institute of MGH, MIT and Harvard, Boston, MA 02114, USA
18
[Abstract Word Count = 172
Text Word Count = 5,307]
19
Corresponding Author:
Dan H. Barouch
20
Address:
Center for Virology and Vaccine Research
21
Beth Israel Deaconess Medical Center
22
E/CLS-1047, 330 Brookline Avenue
23
Boston, MA 02215, USA
Downloaded from http://jvi.asm.org/ on May 22, 2018 by guest
4
24
E-mail:
[email protected]
25
Tel No:
(617) 735-4485
26
Fax No:
(617) 735-4566
27
Running Title: Long-Term HIV-1 Env Vaccine in Guinea Pigs
28
Keywords: HIV-1, vaccine, neutralizing antibodies, long-term, multivalent, gp140
Downloaded from http://jvi.asm.org/ on May 22, 2018 by guest
29
ABSTRACT A vaccination regimen capable of eliciting potent and broadly neutralizing antibodies
31
(bNAbs) remains an unachieved goal of the HIV-1 vaccine field. Here we report the
32
immunogenicity of longitudinal prime/boost vaccination regimens over a period of 200 weeks in
33
guinea pigs with a panel of HIV-1 envelope (Env) gp140 protein immunogens. We assessed
34
vaccine regimens that included a monovalent clade C gp140 regimen (C97), a tetravalent
35
regimen consisting of four clade C gp140s (4C), and a tetravalent regimen consisting of a clade
36
A, B, C, and mosaic gp140 (ABCM). We found that the 4C and ABCM prime/boost regimens
37
were capable of eliciting a greater magnitude and breadth of binding antibodies targeting
38
variable loop 2 (V2) over time, compared to the monovalent C97 only regimen. The longitudinal
39
boosting regimen conducted over more than two years increased the magnitude of certain tier 1
40
NAbs, but did not increase the magnitude or breadth of heterologous tier 2 NAbs. These data
41
suggest that additional immunogen design strategies are needed to induce broad, high titer tier 2
42
NAbs.
Downloaded from http://jvi.asm.org/ on May 22, 2018 by guest
30
43
IMPORTANCE The elicitation of potent, broadly neutralizing antibodies (bNAbs) remains an elusive goal
45
for the HIV-1 vaccine field. In this study we explored the use of a long-term vaccination regimen
46
with differing immunogens to determine if we could elicit bNAbs in guinea pigs. We found that
47
longitudinal boosting over more than two years increased tier 1 NAbs but did not increase the
48
magnitude and breadth of tier 2 NAbs. These data suggest that additional immunogen designs
49
and vaccination strategies will be necessary to induce broad tier 2 NAb responses.
Downloaded from http://jvi.asm.org/ on May 22, 2018 by guest
44
50
INTRODUCTION Successful elicitation of broadly neutralizing antibodies (bNAbs) against the HIV-1
52
envelope (Env) protein through vaccination remains an important but unachieved goal. It is
53
known that 15-20% of individuals chronically infected with HIV-1 are capable of eliciting
54
bNAbs (1-4). These individuals first develop NAbs (5, 6), which drive viral escape and
55
evolution, resulting in expansion of Env diversity (4, 7-9). In some patients, this sequence
56
diversity drives the development of bNAbs capable of targeting conserved epitopes (10-13).
57
These studies suggest that the long-term exposure of the immune system to multiple diverse Env
58
sequences diversity can result in the development of bNAbs.
59
No HIV-1 vaccine to date has been capable of eliciting bNAbs in humans (14-17). A
60
variety of strategies have been explored with the goal of expanding the breadth of vaccine-
61
elicited NAbs. One strategy assessed mixtures of different Envs with the goal of exposing B cells
62
to sequence diversity, but failed to improve the breadth of tier 2 NAb responses (18-23).
63
Additionally, groups have utilized rationally designed immunogens focused on eliciting bNAbs
64
to a single Env epitope, such as the CD4 binding site, but have not driven the full development of
65
such bNAbs (24-26). Mimics of the native HIV-1 Env trimer, such as the SOSIP trimer, have
66
also been assessed, but elicited NAbs to a limited breadth of tier 2 pseudoviruses and targeted a
67
hole in the glycan shield (27-30). Finally, long-term vaccination strategies have been considered
68
with the goal of allowing for affinity maturation and the development of neutralization breadth.
69
Few studies have explored vaccination regimens spanning multiple years, however, they have
70
also failed to induce tier 2 neutralization breadth (31).
71
In this study, we evaluated the effects of a longitudinal prime/boost vaccination regimen
72
on the evolution of binding and NAb responses in guinea pigs over a vaccination regimen that
Downloaded from http://jvi.asm.org/ on May 22, 2018 by guest
51
spanned more than two years. We found that multivalent, sequential prime/boost vaccination
74
regimens improved the breadth of binding antibodies compared to vaccination with a single Env.
75
Additionally, while we observed a limited breadth of tier 2 NAbs in all vaccination regimens, the
76
breadth and magnitude of these NAbs did not increase over the course of the longitudinal
77
regimen. These data suggest that novel immunogen design strategies and vaccination regimens
78
will be needed to improve tier 2 NAb responses.
Downloaded from http://jvi.asm.org/ on May 22, 2018 by guest
73
79
MATERIALS AND METHODS
80
Plasmids, Cell Lines, Protein Production, and Antibodies
81
The codon-optimized synthetic genes of all HIV-1 Env gp140s were produced by
82
GeneArt (Life Technologies). All constructs contained a consensus leader signal sequence
83
peptide, as well as a C-terminal foldon trimerization tag followed by a His-tag (32, 33). All gp140 proteins were expressed in 293T cells utilizing stable cell lines (C97ZA012,
85
92UG037, Mosaic 3.1) (Codex Biosolutions) or transient transfections with polyethylenimine
86
(405C, 459C, 939C, PVO.4) (34-36). His-tagged proteins were purified by HisTrap Ni-NTA
87
column (GE Healthcare). Ni-NTA columns were washed with 20 mM imidazole (pH 8.0) and
88
protein was eluted with 300 mM imidazole (pH 8.0). Fractions containing protein were pooled
89
and concentrated. Protein constructs were further purified utilizing gel filtration chromatography
90
on Superose 6 (GE Healthcare) in running buffer containing 25 mM Tris (pH 7.5) and 150 mM
91
sodium chloride. Purified proteins were concentrated using CentriPrep YM-50 concentrators
92
(Millipore), flash frozen in liquid nitrogen, and stored at -80C.
93
3BNC117 was provided by Michel Nussenzweig (Rockefeller University, New York,
94
NY). PG9 and 447-52D were purchased from Polymun Scientific. PGT121 was purchased from
95
Catalent. Gp70 V1/V2 HIV-1 envelope scaffolds including ConC, Case A2, CN54, and A244
96
V1/V2 were purchased from Immune Technology Corp.
97
Guinea Pig Vaccinations
98
Outbred female Hartley guinea pigs (Elm Hill) were used for all vaccination studies and
99
were housed at the Animal Research Facility of Beth Israel Deaconess Medical Center under
100
approved Institutional Animal Care and Use Committee (IACUC) protocols.
Downloaded from http://jvi.asm.org/ on May 22, 2018 by guest
84
Guinea pigs (n=5/group) were immunized with Env protein intramuscularly in the
102
quadriceps bilaterally at 4-week intervals, with two long term rests, for a total of 12 injections.
103
Vaccination groups included (1) C97ZA012 gp140 only (C97, n=5); a sequential prime/boost
104
regimen including a clade C C97ZA012, a clade A 92UG037, Mosaic 3.1, and clade B PVO.4
105
gp140s (ABCM, n=5); and clade C C97ZA012, 459C, 405C, and 939C gp140s (4C, n=5) (Figure
106
1A). For ABCM, one animal died following blood draw and was excluded from the analysis.
107
One guinea pig passed at week 134 and one at week 177 in the C97 vaccination group due to
108
age.
109
12 vaccinations were given as three sets of 4 immunizations at weeks 0, 4, 8, 12, at weeks
110
62, 66, 70, and 74, and at weeks 104, 108, 112, 116 (Figure 1A). Animals were followed for a
111
total of 200 weeks. Vaccinations consisted of a total of 100 g of Env gp140 per injection
112
formulated in 15% Emulsigen (vol/vol) oil-in-water emulsion (MVP Laboratories) and 50 g
113
CpG (Midland Reagent Company) as adjuvants. Serum samples were obtained from the vena
114
cava of anesthetized animals.
115
Endpoint ELISAs
116
Serum binding antibodies against gp140 and V1/V2 scaffolds were measured by endpoint
117
enzyme-linked immunosorbant assays (ELISAs) (33). Briefly, ELISA plates (Thermo Scientific)
118
were coated overnight with individual gp140s or V1/V2 scaffolds and incubated overnight. For
119
isotype and subclass ELISAs, plates were coated with C97ZA012 gp140. Guinea pig sera were
120
then added in serial dilutions and detected with an HRP-conjugated goat anti-guinea pig
121
secondary antibody for total IgG ELISAs (Jackson ImmunoResearch Laboratories). For
122
isotyping ELISAs, HRP-conjugated goat anti-guinea pig IgG1, goat anti-guinea pig IgG2, goat
123
anti-guinea pig IgM, and sheep anti-guinea pig IgA were utilized (MyBiosource). Plates were
Downloaded from http://jvi.asm.org/ on May 22, 2018 by guest
101
124
developed and read using the Spectramax Plus ELISA plate reader (Molecular Devices) and
125
Softmax Pro-4.7.1 software. End-point titers were considered positive at the highest dilution that
126
maintained an absorbance >2-fold above background values.
127
Avidity ELISAs Serum binding antibody avidities to HIV-1 Env gp140 were measured by a urea
129
disruption enzyme-linked immunosorbent assay (ELISA) (37). Briefly, ELISA plates (Thermo
130
Scientific) were coated with individual Env gp140s and incubated overnight. Guinea pig sera
131
were prepared in a dilution plate to an OD450-OD800 nm absorbance between 1.0 and 1.5 to
132
provide antibody concentrations within a linear range. Sera was plated in duplicate twice; one
133
duplicate for treatment with 8 molar urea (Sigma Aldrich); the other treated with distilled water
134
as a control. Plates were then incubated with an HRP-conjugated goat anti-guinea pig secondary
135
antibody (Jackson ImmunoResearch Laboratories). Plates were developed and read using the
136
Spectramax Plus ELISA plate reader (Molecular Devices) and Softmax Pro-4.7.1 software
137
(optical density 450 – optical density 800). Percent avidity was calculated as [(average
138
absorbance urea treated sample/average absorbance water treated, matched sample)*100]. The
139
avidity index describes 0-30% as low avidity, 30-50% as moderate avidity, and >50% as high
140
avidity binding antibodies.
141
Competition ELISAs
142
PG9, 3BNC117, PGT121, and 447-52D IgG were biotinylated using EZ-Link Micro
143
NHS-PEG4-Biotinylation Kit (Thermo Scientific) following manufacturer’s instructions.
144
Antibodies were used at an OD450-OD550 nm reading of approximately 1.0-1.5 for each coating
145
protein. For PG9 IgG competition, Mosaic 3.1 Env gp140 was utilized as a coating protein and
146
for 3BNC117, PGT121, and 447-52D IgG competition, C97ZA012 Env gp140 was utilized as a
Downloaded from http://jvi.asm.org/ on May 22, 2018 by guest
128
coating protein. ELISA plates (Thermo Scientific) were coated overnight with gp140s. Guinea
148
pig sera added in blocking buffer at a 1:10 dilution and serially diluted at a 1:3 dilution down the
149
plate. Week 0, 16, 78, 120, 138, and 200 samples were run side-by-side on the same plate. A
150
single biotinylated, monoclonal IgG was then added at a single concentration. Streptavidin-HRP
151
(ThermoScientific) in blocking buffer is then added. Plates were then developed with SureBlue
152
tetramethylbenzidine (TMB) Microwell Peroxidase Substrate and TMB Stop Solution
153
(Kirkegaard & Perry Laboratories, Inc.). Plates were developed and read at OD450-OD550 nm
154
using the Spectramax Plus ELISA plate reader (Molecular Devices) and Softmax Pro-4.7.1
155
software.
156
Peptide Microarrays
157
Peptide arrays were generated, conducted, and analyzed using methods as described
158
previously (38). RepliTope Antigen Collection HIV Ultra slides (JPT Peptide Technologies
159
GmbH) were utilized. Each slide contains 6,654 15-mer peptides, printed in triplicate
160
(subarrays), overlapping by 11 amino acids, representing 135 different clades or circulating
161
recombinant forms (CRFs) that cover the entire HIV-1 genome. Microarray slides were
162
incubated with guinea pig sera diluted 1/200 in SuperBlock T20 (TBS) Blocking Buffer (Thermo
163
Scientific). Alexa Fluor 647-conjugated AffiniPure Goat Anti-Guinea Pig IgG (H+L) was used
164
as the secondary detection antibody (Jackson ImmunoResearch Laboratories). All batches of
165
slides with the same time point were run in parallel with a control slide incubated with the
166
secondary antibody only.
167
Slides were scanned with a GenePix 4300A scanner (Molecular Devices), using 635 nm
168
and 532 nm lasers. The fluorescent intensity for each feature (peptide spot) as well as threshold
169
values for positivity were calculated using GenePix Pro 7 software and GenePix Array List as
Downloaded from http://jvi.asm.org/ on May 22, 2018 by guest
147
described previously (38, 39). A control slide incubated with just the secondary fluorophore was
171
subtracted from all experimental slides to remove background associated with the fluorophore.
172
For each batch of slides run together, the highest P
