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pubs.acs.org/NanoLett Letter

Improvement of B Cell Responses by an HIV‑1 Amphiphilic Polymer

Nanovaccine
Xiaoqian Xin, Yifeng Liu, Lei Guo, Hui Wang, Daiqiang Lu, Yaotian Chang, Mingming Wan,
Yong Zhang, Yaming Shan, Qiao Zhang, Xiaowen Liu,* and Feng Gao*
Cite This: https://doi.org/10.1021/acs.nanolett.3c01241 Read Online

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ABSTRACT: The human immunodeficiency virus (HIV) has

infected over 84 million people since its discovery and is a huge
threat to human health. While an HIV vaccine is urgently needed
to curb this devastating pandemic, it has been notoriously difficult
Downloaded via 139.193.206.231 on May 2, 2023 at 06:32:47 (UTC).

to develop, partly due to the extraordinary high level of genetic

variation of HIV. We designed a new HIV-1 envelope glycoprotein
nanoparticle (Env/NP) vaccine using amphiphilic polymers. The
Env/NP vaccine induced more potent and broader neutralizing
activities against multiple HIV-1 subtypes. Moreover, it elicits
similar neutralizing antibody responses after the storage at −80 °C,
4 °C or room temperature post lyophilization. These results
demonstrate that the new Env/NP vaccine not only improves the
HIV vaccine immune responses but also is stable under different storage conditions. This new nanovaccine approach can readily
apply to other protein-based vaccines.
KEYWORDS: amphiphilic polymers, HIV-1, envelope glycoprotein, nanovaccine, neutralization, stability

A cquired immunodeficiency syndrome (AIDS) is a

grievous infectious disease caused by infection of the
human immunodeficiency virus (HIV). By the end of 2021,
approximately 84.2 million people have become infected with
HIV.1 Although the combination antiretroviral therapy
(cART), which uses multiple drugs in the same treatment
regimen, effectively suppresses HIV replication and has
significantly improved the life span and quality of AIDS
patients,2,3 long-term medications have side effects and are
prone to develop drug resistance. Therefore, cART alone will
not end the HIV pandemic. The development of a safe and
effective vaccine is critically needed to control this devastating
AIDS pandemic. However, due to the extraordinary high level
of genetic variation and lack of the fundamental understanding
of immune protection mechanisms, all HIV vaccine efficacy
trials have failed except the RV144 trial, which showed a
modest efficacy.4,5
Recently, native-like HIV-1 envelope (Env) glycoprotein
trimers such as soluble, stabilized, proteolytically cleaved,
trimeric forms of Env (SOSIP gp140 proteins),6 native flexibly
linked (NFL) trimers,7 and uncleaved prefusion-optimized
(UFO) trimers8 have been developed and have induced better
neutralizing antibody (nAb) responses than the monomer Env
immunogens in guinea pigs, rabbits, and monkeys.9−11
However, they only elicit nAbs against autologous hard-to-
neutralize tier 2 viruses.11 Such immune responses generally
require prolonged, multiple immunizations and rely on
adjuvants to boost the immune responses. Thus, exploring
innovative nanomaterials to enhance immune responses of
protein antigens and increase the resistance to hash storage
conditions will assist in developing potent and effective HIV-1
vaccines.
Herein, we synthesized the amphiphilic polymer (P1M10) by
conjugating poly(maleic anhydride-ALT-1-octadecene)
(PMHC18) with poly(ethylene glycol) (PEG). P1M10 can
embed proteins through intermolecular forces to form a
nanoformulation. We have shown that P1M10 renders the
embedded proteins stable at a high temperature12,13 and
maintains slow insulin release into blood for a long period of
time.13 Here, we utilize these advantages of P1M10 to improve
the immune responses induced by an HIV-1 vaccine candidate.
We find that the HIV-1 Env nanoparticle (Env/NP) vaccine
generated with P1M10 can enhance the immune responses by
increasing neutralization potency and breadth. In addition,
Env/NP is stable at room temperature (RT) and can ease the
stringent temperature requirements for storage and trans-
portation.
Received: April 2, 2023
Revised: April 17, 2023

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Figure 1. Preparation and characterization of Env/NP. (A) Schematic diagram for preparation of Env/NP. Hydrophilic PEG is tagged to PMHC18
via amidation reaction to form the amphiphilic polymer P1M10, which assembled into NPs together with HIV-1 Env trimers by intermolecular
forces. (B) Loading of the Env trimers into Env/NP. The dips at 1655 cm−1 and 1535 cm−1 correspond to the amide I and amide II groups in Env
trimers, respectively, were detected by FT-IR spectrum. (C) Analysis of the secondary structure of the Env trimers in Env/NP by CD
Spectrometer. (D) Size distribution of the Env trimers and nanoparticles. (E) Morphology of Env/NPs. Env/NPs are shown as irregular spheres
with an average diameter of 20 nm (15−25 nm) as determined by TEM. (F) Determination of surface charges by measuring Zeta potentials. Data
is shown as the mean ± SD (n = 3). (G) Release rate of Env from Env/NP in PBS at 37 °C. Data is shown as mean ± SD (n = 3). Env trimers,
P1M10, and Env/NP are shown in green, blue, and red, respectively.

P1M10 was prepared by the amidation reaction between the
anhydride group on PMHC18 and the amino group on PEG.
The actual average number of PEGs anchored on PMHC18 was
determined by 1H NMR.14 The characteristic absorption peaks
of PMHC18 and PEG were detected at chemical shifts of 1.1−
1.3 ppm and 3.8−3.5 ppm, respectively (Figure S1),15,16
indicating that PMHC18 and PEG were successfully con-
jugated. The amphipath of P1M10 allowed it to bind to the
hydrophobic, hydrophilic, or positively charged moieties of
HIV-1 Env trimer surface to form Env/NP through
intermolecular forces (Figure 1A). Comparison of trans-
mittances of amide I at 1655 cm−1 and amide II at 1535
cm−1 detected by Fourier-transform infrared spectroscopy
(FT-IR) can determine whether a protein is embedded into
nanoparticles.17 The similar transmittance curves of amide I
and amide II detected in both Env trimer and Env/NP
indicated that the Env trimers were successfully embedded into
the nanoparticle and that their structure conformation were
well preserved (Figure 1B). Analysis of the circular dichroism
(CD) signals showed that both the Env trimer and Env/NP
had similar curve changes (Figure 1C). This further confirmed
that the Env secondary structures in Env/NP was well
preserved. To determine the encapsulation efficiency, 0.2 mg
of the Env trimers was used to make Env/NP. The final
reaction was centrifuged and 0.09 mg of the uncaptured free
Env trimers was detected in the supernatant. Thus, about half
of the protein (55%) was successfully embedded into the
nanoparticles. While P1M10 could self-assemble into nano-
particles with the diameters at ∼40 nm due to its amphipath,
B https://doi.org/10.1021/acs.nanolett.3c01241
the diameters of the Env/NP particles decreased to ∼20 nm
when P1M10 bound to HIV-1 Env trimers as determined by
dynamic light scattering (DLS) (Figure 1D). This were further
confirmed by the observation of irregular spheres (∼40 nm) of
P1M10 (Figure S2) and Env/NP spheres (∼20 nm) under
transmission electron microscope observation (Figure 1E).
The surface Zeta potentials of Env trimers, P1M10, and Env/
NP were at −7.6 mV, −1.3 mV, and −1.6 mV (Figure 1F),
respectively. This demonstrated that P1M10 and Env trimers
successfully bound together and significantly reduced the
negative charge of the Env trimers.
To understand the release rate of the Env trimers, Env/NP
was incubated in PBS at 37 °C and the presence of the Env
trimers in the supernatant was monitored. At the beginning
(hour 0), no protein was detected. At hour 1, 7% of the total
embedded protein was released and the protein continued to
rapidly release up to 39% of the total embedded protein by
hour 12 (Figure 1G). Thereafter, the release rate was slowed
and only 12% more was released by hour 48 (total 51%). The
Env trimers released from Env/NP showed the similar circular
dichroism profile as that of the original Env trimers (Figure
S3).
To evaluate the immunogenicity of Env/NP, we immunized
New Zealand rabbits in three groups: Env with adjuvant AS03
(Group 1), Env/NP alone (Group 2), and Env/NP with AS03
(Group 3). The blood samples were collected 2 weeks after
each immunization (Figure S4). The titers of HIV-1 Env
specific binding Abs in sera were detected by ELISA. Binding
Ab titers (1:10,944) in Group 3 were higher than those in
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Figure 2. Enhanced immune responses by Env/NP. (A) Induction of HIV-1 specific binding Abs. Rabbits were immunized with Env trimer with
AS03 (Group 1, n = 2), Env/NP alone (Group 2, n = 3), or Env/NP with AS03 (Group 3, n = 3). (B) Induction of HIV-1 neutralizing Abs against
MW965.26. Black arrows represent the time of immunizations. LOD: limit of detection (1:100 and 1:20 for binding Ab and nAb, respectively).

Figure 3. Increased neutralization breadth induced by Env/NP. (A) Evaluation of the neutralization titers against different HIV-1 strains. (B)
Number of tier 2 heterologous viruses neutralized by the sera from immunized animals. LOD: limit of detection (1:20).

Figure 4. Improved stability of HIV-1 Env/NP at different conditions. (A) Induction of HIV-1 specific binding Abs by differently treated
immunogens. Mice were immunized with three differently treated Env/NPs (−80 °C, 4 °C, and room temperature (lyophilized); n = 5). (B)
Determination of the neutralization titers against three HIV-1 tier 1 strains using sera collected after four immunizations. LOD: limit of detection
(1:100 and 1:50 for binding Ab and nAb, respectively).

Groups 1 (1:2,736) or Group 2 (1:2,873) after the third
immunization and maintained at the similar high titers
(1:18,490) after the fourth immunization (Figure 2A). These
results indicated that the combination of Env/NP and AS03 in
Group 3 induced the higher immune responses. We then
determined nAb activities against MW965.26 in the sera after
each immunization and found that Env/NP with AS03 also
induced higher nAb titers (1:275) than the Env with AS03
(1:39) or the Env/NP alone (1:78) after the third
immunization (Figure 2B). The higher nAb titers induced by
the Env/NP with AS03 than the Env with AS03 demonstrated
the improved immune responses by the novel Env/NP vaccine,
indicating the adjuvant effect of the new nanomaterials.
We next investigated neutralization breadth with a panel of
three easy-to-neutralize tier 1 pseudoviruses (MW965.26,
SF162.LS, 92RW020.2) and 17 hard-to-neutralize tier 2
pseudoviruses (398F1, X2278, TRO11, CE0217, CE1176,
25710, CNE8, CNE55, CH119, 246F3, BJOX2000, X1632,
ZM197M, HIV-16936-2, CAP45, WITO4160, SC422661) on
TZM-bl cells. After the fourth immunization, the sera from all
groups neutralized MW965.26 and only one serum from each
C https://doi.org/10.1021/acs.nanolett.3c01241
group neutralized SF162.LS but no sera from any groups
neutralized 92RW020.2 (Figure 3A). However, the neutraliza-
tion titers against these tier 1 viruses in the sera from Group 3
were higher than those in Groups 1 and 2 (Figure 3A). The
sera from both HIV-1 Env/NP vaccine groups (Groups 2 and
3) could neutralize three tier 2 viruses (X2278, TRO11, and
CE0217) with higher nAb titers while the sera from the Env
with AS03 group only neutralized X2278 (Figure 3A and Table
S1). Overall, the sera from the Env with AS03, Env/NP alone,
and Env/NP with AS03 groups neutralized 1, 4, and 3 tier 2
viruses (Figure 3B and Table S2), respectively, indicating that
the HIV-1 Env/NP vaccine can induce broader nAb responses.
One disadvantage of protein-based vaccines is the cold chain
requirement for storage and transportation.18 Even a short
storage at ambient temperature often results in protein
degradation and significantly reduces vaccine efficacy. In
addition, the cold chain drives up the cost of vaccination by
80%.19 We have previously showed that the amphiphilic
polymers could significantly increase insulin’s resistant to high
temperature and be lyophilized for easy and long-term
storage.13 To investigate whether P1M10 could render the
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HIV-1 Env/NP vaccine more resistant to hash temperature
conditions for convenient storage, the Env/NP vaccine was
either stored at 4 °C for 2.5 months or lyophilized and stored
at RT for 2.5 months. Each treated immunogen was then used
to immunize BALB/c mice four times at a 3-week interval
(Figure S5) and the titers of HIV-1 Env specific binding Abs in
sera were then determined. Env/NP that were stored at 4 °C
for 2.5 months or lyophilized induced similar immune
responses as it was stored at −80 °C (Figure 4A). HIV-1
Env specific Ab responses were detected after two immuniza-
tions and continuously boosted after each additional
immunization for all three treated immunogens (−80 °C, 4
°C, and RT). There were no significant differences in Ab titers
(1:561,503, 1:539,780, and 1:266,501; p > 0.05 among all
three storage conditions) after the fourth immunization among
all three treated immunogens (Figure 4A). We then
determined nAb activities against three tier 1 pseudoviruses
(MW965.26, SF162.LS, and 92RW020.2) using the sera after
four immunizations. Sera from mice immunized with three
differently treated immunogens showed high neutralization
titers against MW965.26, low titers against SF162.LS, and
barely detectable titers against 92RW020.2 (Figure 4B). Env/
NP stored at RT (lyophilized) induced similar nAb responses
as Env/NP stored at −80 °C, while Env/NP stored at 4 °C
induced slightly lower bnAb titers than Env/NP stored at −80
°C or RT (lyophilized). However, these differences are not
statistically significant (p > 0.05 among all three storage
conditions). These results show that all three differently
treated Env/NPs (−80 °C, 4 °C, and RT) induced comparable
binding and neutralizing Ab titers, indicating that the HIV-1
Env/NP vaccine can be easily stored and transported at
ambient temperature after lyophilization although more hash
conditions like higher temperature and longer storage time
need to be tested.
We have developed a new nanoparticle vaccine based on the
amphiphilic polymer P1M10. There are three advantages for
this vaccine. First, it enhances immune responses compared to
the native-like Env trimers by increasing the nAb titers.
Second, it increases the neutralization breadth against different
HIV-1 strains. Third, it exhibits excellent stability and can be
lyophilized for long-term storage at ambient temperature,
which could significantly ease the stringent temperature
requirements for storage and transportation of HIV-1 Env
vaccines. Importantly, this new nanoparticle vaccine approach
can be applied to other protein-based vaccines to further
improve their immunogenicity and render them less dependent
on the cold chain storage and transportation.
■
*
ASSOCIATED CONTENT
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.nanolett.3c01241.
Additional information on experimental materials and
methods including figures for 1H NMR spectra analysis
of P1M10, immunization procedure and tables for
analysis of the neutralization activity in the sera and
comparison of the neutralization breadth among differ-
ent immunogens (PDF)
D https://doi.org/10.1021/acs.nanolett.3c01241
Letter
■ AUTHOR INFORMATION
Corresponding Authors
Xiaowen Liu − Engineering Center of Catalysis and Synthesis
for Chiral Molecules, Department of Chemistry, Fudan
University, Shanghai 200433, China; Shanghai Engineering
Research Center of Industrial Asymmetric Catalysis of Chiral
Drugs, Shanghai 200433, China; Email: liuxw@
fudan.edu.cn
Feng Gao − Institute of Molecular and Medical Virology, Key
Laboratory of Ministry of Education for Viral Pathogenesis &
Infection Prevention and Control, School of Medicine, Jinan
University, Guangzhou, Guangdong Province 510632, China;
Email: fenggao@jnu.edu.cn
Authors
Xiaoqian Xin − Institute of Molecular and Medical Virology,
Key Laboratory of Ministry of Education for Viral
Pathogenesis & Infection Prevention and Control, School of
Medicine, Jinan University, Guangzhou, Guangdong Province
510632, China; orcid.org/0000-0002-2460-0989
Yifeng Liu − Institute of Molecular and Medical Virology, Key
Laboratory of Ministry of Education for Viral Pathogenesis &
Infection Prevention and Control, School of Medicine, Jinan
University, Guangzhou, Guangdong Province 510632, China
Lei Guo − Institute of Molecular and Medical Virology, Key
Laboratory of Ministry of Education for Viral Pathogenesis &
Infection Prevention and Control, School of Medicine, Jinan
University, Guangzhou, Guangdong Province 510632, China
Hui Wang − Institute of Molecular and Medical Virology, Key
Laboratory of Ministry of Education for Viral Pathogenesis &
Infection Prevention and Control, School of Medicine, Jinan
University, Guangzhou, Guangdong Province 510632, China
Daiqiang Lu − Institute of Molecular and Medical Virology,
Key Laboratory of Ministry of Education for Viral
Pathogenesis & Infection Prevention and Control, School of
Medicine, Jinan University, Guangzhou, Guangdong Province
510632, China
Yaotian Chang − National Engineering Laboratory for AIDS
Vaccine, School of Life Sciences, Jilin University, Changchun,
Jilin Province 130012, China
Mingming Wan − National Engineering Laboratory for AIDS
Vaccine, School of Life Sciences, Jilin University, Changchun,
Jilin Province 130012, China
Yong Zhang − National Engineering Laboratory for AIDS
Vaccine, School of Life Sciences, Jilin University, Changchun,
Jilin Province 130012, China
Yaming Shan − National Engineering Laboratory for AIDS
Vaccine, School of Life Sciences, Jilin University, Changchun,
Jilin Province 130012, China; orcid.org/0000-0001-
8373-6088
Qiao Zhang − Institute of Molecular and Medical Virology,
Key Laboratory of Ministry of Education for Viral
Pathogenesis & Infection Prevention and Control, School of
Medicine, Jinan University, Guangzhou, Guangdong Province
510632, China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.nanolett.3c01241
Author Contributions
F.G., X.L., and X.X. designed the study; F.G., X.L., and Q.Z.
jointly supervised the study; X.X. performed experiments and
analyzed the results; Y.L., L.G., H.W, and Q.L. assisted in
animal experiments and neutralization assays; Y.C., Y.Z., and
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Nano Letters pubs.acs.org/NanoLett Letter

Y.S. generated Env trimers; X.X. and F.G. wrote the paper. All
authors have reviewed and approved the paper.
Notes
The authors declare no competing financial interest.
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■ ACKNOWLEDGMENTS
We thank Jiang Zhu for the gift of plasmids expressing HIV-1
Env trimer. This work was supported by the National Key
Research and Development Program of China
2021YFC2301500.
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Black Phosphorus Anodes. ACS Appl. Mater. Interfaces 2021, 13 (11),
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Qi, L.; Wu, D.; Xu, Y.; et al. Rapid synthesis of ‘yolk-shell’-like
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