The Effects of Heat Shock Proteins on the Expression of HIV-1 Nef
Antigen in Mammalian Cells
Alireza Milani1 , Fatemeh Rouhollah1, Maryam Naseroleslami1 , Azam Bolhassani2*
1

Department of Cellular and Molecular Biology, Faculty of Advanced Sciences and Technology, Tehran Medical Sciences,
Islamic Azad University, Tehran, Iran; 2Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, IranART IC LE INFO
Original Article
VacRes, 2020
Vol. 7, No. 1, 54-59
Received: August 23, 2020
Accepted: September 23, 2020
Pasteur Institute of Iran

[ DOI: 10.29252/vacres.7.1.54 ][ Downloaded from vacres.pasteur.ac.ir on 2024-10-19
2023-12-18 ]*Corresponding Author:
Azam Bolhassani, PhD, Department of
Hepatitis and AIDS, Pasteur Instituteof Iran, Tehran, Iran.
Email: azam.bolhassani@yahoo.com
Tel/Fax: (+98)2166953311 Ext. 2240/(+98)2166465132
KEYWORDS: HIV-1, Nef Antigen,
Heat Shock Protein, Hsp27, Hsp70,
DNA Vaccine

ABSTRACTIntroduction: Vaccine design is mainly considered as a therapeutic strategy to elicit HIVspecific immunity. DNA vaccines encoding an antigen and also an adjuvant can induce aneffective adaptive immunity. Due to having numerous roles in viral infection, the HIV-1
Nef protein is considered as an antigen candidate for development of therapeutic vaccines.
A variety of adjuvants and delivery systems have been utilized to increase the potency of
DNA vaccines against viral infections, such as heat shock proteins (HSPs) which possesschaperon activity and immunostimulatory properties. Methods: pEGFP mammalian
expression vectors harboring nef, hsp27, hsp27-nef, hsp70 and hsp70-nef genes wereprepared in large scale. Their concentration and purity were assessed by NanoDrop
spectrophotometry. Human embryonic kidney 293T cells (HEK-293T) were grown in
DMEM culture medium and transfected with these constructs using Lipofectamine 2000
transfection reagent. After 48 hours, their transfection efficiency was evaluated using
fluorescent microscopy and flow cytometry. Results: The pEGFP-nef, -hsp27, -hsp27-nef,-hsp70 and -hsp70-nef constructs were successfully prepared in large scale and highpurity. The results of cell transfection with each construct showed that the percentages of
Nef-GFP, Hsp27-GFP, Hsp27-Nef-GFP, Hsp70-GFP, Hsp70-Nef-GFP, Hsp70-GFP +Nef-GFP and GFP expression were 53.1  0.2, 64.22  0.8, 57.1  0.7, 68.8  1.0, 61.7 
0.7, 77.4  1.5 and 81.8  1.8, respectively. Conclusion: These data showed that thepotency of Hsp70 was more than Hsp27 for increasing Nef-GFP expression in HEK-293T
cells. Moreover, the delivery of pEGFP-nef along with pEGFP-hsp70 increased the rate of
GFP population in the cells as compared to that in pEGFP-hsp70-nef. These constructs
will be used for development of HIV-1 DNA vaccine in animal model in near Future.
Citation:
Milani A, Rouhollah F, Naseroleslami M, Bolhassani A. The Effects of Heat Shock Proteins on
Delivery of HIV-1 Nef Antigen in Mammalian Cells. vacres. 2020; 7 (1) :54-59. DOI:
10.29252/vacres.7.1.54

INTRODUCTION
Designing an effective vaccine against humanimmunodeficiency virus (HIV) has remained as an intractablechallenge [1-3]. Some evidences confirmed the importance ofvirus-specific cellular immune responses in controlling HIV-1
replication; thus different therapeutic vaccine strategies basedon viral or bacterial vectors, DNA, RNA, peptide and protein
are being developed in preclinical or clinical trials [4]. It has
been determined that DNA vaccination can effectively
stimulate both innate and adaptive immunities (e.g. CD8+ T
cell and CD4+ T helper cell immune responses) [5]. The
plasmid DNA encoding a therapeutic antigen can be directly
designed based on pathogen sequences, allowing flexibility andspeed in preclinical testing and subsequently rapid transition toclinical scale up [6]. Among the HIV-1 genes, the nef
regulatory gene plays a critical role in viral infection including

down-regulation of cell surface receptors such as MHC-I and
CD4, enhancement of viral replication, and alteration of T cell
activation. Also, Nef can be considered as an attractive antigen
candidate in therapeutic vaccine development due to its
multiple immunogenic epitopes [7-9)]. Practical considerationsof DNA vaccines as an alternative to other therapeutic methods
against HIV depends on the improvement of delivery systems
and also adjuvants. The endogenous adjuvants such as heatshock proteins (HSPs) has been suggested to induce antigenspecific humoral and cellular immunity, effectively [10].
Among HSPs, small heat shock protein 27 (Hsp27) can
modulate the immune system through interaction with toll likereceptors-2 and -4 [11]. Hsp70 could increase the presentationand cross-presentation of antigens to the immune system fordevelopment of innovative therapeutic vaccines against cancers

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The Effects of Heat Shock Proteins on Delivery of HIV-1 Nef Antigen in Mammalian Cells

and chronic viral infections [12]. To develope DNA vaccine, in
vitro expression of the gene constructs could facilitate rapid
screening and selection of potential vaccine candidates. Herein,
we prepared DNA constructs of Nef, Hsp27, Hsp27-Nef, Hsp70
and Hsp70-Nef and evaluated their expression in a mammaliancell line. These constructs were transfected to the HEK-293T
cell line using Lipofectamine 2000 transfection reagents andtheir expression was assessed by fluorescent microscopy andflow cytometry.

fluorescent protein (EGFP) were employed in this study. The
constructions of the full length pEGFP-nef, pEGFP-hsp27 andpEGFP-hsp27-nef genes were described in our previous study[13]. Moreover, the full length of hsp70 gene was prepared in a
pUC57 cloning vector by Biomatik Company (Canada). To
generate pEGFP-hsp70, the fragment was subcloned into
pEGFP-N3 eukaryotic expression vector in NheI/ SalI sites(Fermentas) (Fig. 1.A). Also, to make pEGFP-hsp70-nef, thenef fragment was amplified by PCR from pUC19-nef using
primers designed to generate EcoRI and SalI restriction sites at
5’ and 3’ ends of the amplified fragment, as shown below.
Then, the nef gene was ligated in a linearized pEGFP-hsp70
between EcoRI and SalI sites using T4 DNA Ligase(Fermentas) (Fig. 1.B).

MATERIALS and METHODS[ DOI: 10.29252/vacres.7.1.54 ][ Downloaded from vacres.pasteur.ac.ir on 2024-10-19
2023-12-18 ]Preparation of the Recombinant Plasmids
To monitor the efficiency of transfection, pEGFP-N1 andpEGFP-N3 eukaryotic vectors encoding enhanced green

Fig. 1. Schematic diagram for the construction of pEGFP-hsp70 and pEGFP-hsp70-nef. (A) pEGFP-hsp70: The hsp70
fragment was digested from pUC57-hsp70 with NheI/SalI and subcloned into the pEGFP-N3 eukaryotic vector. (B) pEGFPhsp70-nef: The PCR product of nef gene was digested with EcoRI/SalI and cloned into the recombinant pEGFP-Hsp70 vector.

Forward primer: 5’-CGGAATTCATGGGTGGCSSGTGGTC-3’ (EcoRI)
Reverse primer: 5’-GGATTCGTCGACGCAGTTCTTGAAGTAC-3’ (SalI)

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The Effects of Heat Shock Proteins on Delivery of HIV-1 Nef Antigen in Mammalian Cells

After transformation of E.coli DH5α strain with the ligatedsolution, the recombinant plasmids were purified from clones
using plasmid DNA extraction mini-kit (FavorPrepTM, Taiwan)
according to manufacturer’s instructions. The concentration andpurity of plasmid DNA were determined by NanoDrop™ Lite
Spectrophotometer (Thermo Fisher Scientific).

RESULTS
Generation of the Recombinant DNA Constructs
The subcloning of hsp70 gene into pEGFP-N3 vector was
confirmed by the presence a ~1950 bp fragment on 1% gelelectrophoresis. Moreover, a clear band of ~2600 bp for the
hsp70-nef fusion was observed on the gel after NheI/SalI
double-digestion as shown in Fig. 2.

[ DOI: 10.29252/vacres.7.1.54 ][ Downloaded from vacres.pasteur.ac.ir on 2024-10-19
2023-12-18 ]Cell Culture
Human Embryonic Kidney 293T (HEK-293T) cell line
(Pasteur institute of Iran) were grown in Dulbecco’s Modified
Eagle’s Medium (DMEM; Sigma), supplemented with 10%
fetal bovine serum (FBS, Gibco), pen/strep (100U/ml penicillin
and 0.1 mg Streptomycin; Gibco). The cells were seeded at
5×104 cells/well in 24-well plates , approximately 24 hours
prior to transfection and incubated under standard cell culture
conditions (37°C, 5% CO2, humidified air) for achieving
approximately 80% confluency.

Transfection
The plasmid DNAs, namely pEGFP-N1, pEGFP-nef,
pEGFP-hsp27, pEGFP-hsp27-nef, pEGFP-hsp70 and pEGFPhsp70-nef were transfected using LipofectamineTM 2000
(Invitrogen, USA) based on the manufacturer’s protocols. Also,
the cells were cotransfected with pEGFP-nef + pEGFP-hsp70
for comparison with transfection of pEGFP-hsp70-nef
construct. For production of lipofectamine/ plasmid DNA
complex, 50 µl of serum-free DMEM was mixed with 2 µl of
lipofectamine and incubated for 5 min in room temperature.
Then, 50 µl incomplete DMEM was mixed with 1 µg of eachplasmid DNA and added to lipofectamine solution. The
mixtures were incubated for 30 min in room temperature toform the DNA/lipofectamine complexes. The complexes werethen added to each well and the medium was replaced after 5 hwith pre-warmed complete DMEM (5% FBS). Finally, theplates were incubated for 48 hours at 37˚C for measuringtransient transfection efficiency. The cells transfected with
pEGFP-N1 vector were used as a positive control. The
untreated cells were considered as a negative control.

Fig. 2. Confirmation of the recombinant plasmids by
double-digestion on gel electrophoresis.
Lanes 1 and 2 represent the double digested products using
NheI/SalI restriction enzymes for pEGFP-hsp70 (~ 1950 bp)
and pEGFP-hsp70-nef (~ 2600 bp), respectively.

The nef, hsp27 and hsp27-nef genes were previously
confirmed as clear bands of ~648 bp, ~720 bp and ~1368 bp on
agarose gel [13]. The pEGFP-nef, pEGFP-hsp27 and pEGFPhsp27-nef, pEGFP-hsp70 and pEGFP-hsp70-nef were preparedwith high purity.

In Vitro Transfection Assay
Fluorescent microscopy and flow cytometry were used formonitoring the efficiency of plasmid DNA uptake into the cells
using GFP reporter. The expressions of Nef-GFP, Hsp27-GFP,
Hsp27-Nef-GFP, Hsp70-GFP, Hsp70-Nef-GFP and GFP
proteins were detected by fluorescent microscopy (Envert
Fluorescent Ceti, Korea) and quantified by a flow cytometer(Partec, Germany) after 48 hours cell transfection. For flow
cytometry analysis, the cells were harvested by trypsin 1X andthe cell pellets were resuspended in PBS 1X (pH 7.4). The
expressions of the fluorescent genes were measured in FL1channel using an excitation filter (485 nm) and an emission
filter (535 nm).

Evaluation of Transfection Efficiency Using Fluorescent
Microscopy and Flow Cytometry
The DNA transfection efficiency using Lipofectamine was
detected by fluorescent microscopy and flow cytometry. These
results were determined by the percentage of protein expression
using GFP reporter. The percentage of Nef-GFP and GFP
expression were 53.1  0.2 and 81.8  1.8, respectively (Fig. 3).
The transfection rates of Hsp27-encoding DNA constructs
were 64.22  0.8 and 57.1  0.7 for Hsp27-GFP and Hsp27Nef-GFP, respectively. Also, the transfection percentages ofHsp70-encoding DNA constructs were 68.8  1.0, 61.7  0.7
and 77.4  1.5 for Hsp70-GFP, Hsp70-Nef-GFP and Hsp70GFP + Nef-GFP, respectively. The transfection efficiency of
Hsp-encoding constructs was shown in Fig. 4.

Statistical Analysis
Prism 8.3 software (GraphPad, San Diego, California,
USA) was used for statistical analysis. The differences between
the control and the test groups were assessed using one-way
ANOVA (GraphPad Prism, GraphPad Software) wherep < 0.05 was considered statistically significant.

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The Effects of Heat Shock Proteins on Delivery of HIV-1 Nef Antigen in Mammalian Cells[ DOI: 10.29252/vacres.7.1.54 ][ Downloaded from vacres.pasteur.ac.ir on 2024-10-19
2023-12-18 ]Fig.3. Analysis of Nef-GFP and GFP expression in HEK-293T cells by Lipofectamine 2000 transfection reagent using fluorescent
microscopy and flow cytometry. The pEGFP-N1 was used as a positive control. The transfection rates of Nef-GFP and GFP expression
were 53.1  0.2 and 81.8  1.8, respectively.

Fig. 4. Analysis of gene expression in HEK-293T cells by Lipofectamine 2000 transfection reagent using fluorescent microscopy and flow
cytometry. The transfection rates of Hsp27-encoding DNA constructs were 64.22  0.8 and 57.1  0.7 for Hsp27-GFP and Hsp27-Nef-GFP,
respectively. Also, the transfection percentages of Hsp70-encoding DNA constructs were 68.8  1.0, 61.7  0.7 and 77.4  1.5 for Hsp70-GFP,
Hsp70-Nef-GFP and Hsp70-GFP + Nef-GFP, respectively.

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The Effects of Heat Shock Proteins on Delivery of HIV-1 Nef Antigen in Mammalian Cells[ DOI: 10.29252/vacres.7.1.54 ][ Downloaded from vacres.pasteur.ac.ir on 2024-10-19
2023-12-18 ]Flow cytometry analysis indicated that the cell penetrationof pEGFP-hsp70-nef using Lipofectamine transfection reagent
was more than pEGFP-hsp27-nef (p < 0.05). Also, theexpression of Hsp70-GFP was more than Hsp27-GFP (p <0.05); thus Hsp70 could increase Nef-GFP expression in thecells, significantly higher than Hsp27 (p < 0.05). Also, thedelivery of pEGFP-nef along with pEGFP-hsp70 increased therate of GFP population in the cells indicating high transfection
efficiency using lipofectamine in HEK-293T cells. The
transfection efficiency is summarized in Fig. 5.

Fig. 5. Comparison of Hsp27 and Hsp70 in delivery of Nef antigen into HEK-293T cells. The expression of the Hsp70-Nef fusion
protein was significantly higher than the Hsp27-Nef fusion protein (p < 0.05). (* p < 0.05, *** p < 0.001, ns: non-significant).

of Nef from T cells to endothelial cells, the Jurkat cells havebeen transfected with HIV-1 Nef, using Lipofectamine LTX
reagent and after 48 hours post transfection, these Jurkat cells
were cocultured with HCAEC cells. The reported data havedemonstrated that the efficiency of Nef transfection was ~20%
in Jurkat cells, and could be transferred from Jurkat toendothelial cells, indicating direct cell-to-cell contact.
Moreover, the efficiency of Nef transfection in HCAEC
endothelial cells has been estimated as ~70% using flow
cytometry [19]. In the recent study, the efficiency of HIV-1 Nef
expression in HEK-293T using Lipofectamine was ~53%.
These differences may be due to the fact that the transfection
efficiency of chemical methods is dependent on the DNA
vector, the reagents and the cell type.
Yamaoka et al. have reported a novel DNA vaccine design
based on Hsp70 and Ovalbumin (OVA) MHC class I epitope
peptide. Before mice immunization with the Hsp70-CTL
epitope fusion construct, this construct was transfected in COS7 cells using Lipofectamine 2000 and its expression was
confirmed by Western blotting [20]. Another study has also
represented transfection of human papillomavirus (HPV)-16
E7-Hsp27 fusion in COS-7 cells using lipofectamine 2000 for
screening potential expression of DNA vaccine candidate ineukaryotic cells [21]. To investigate the heat-shock effect on
gene expression, a pGL3-Hsp70 has been transfected into B16
cells, using Lipofectamine LTX reagent at a ratio of 4:1
reagent/DNA. After 24 hours, the cells were heated at 42.5 °C
for 30 min. Following heat-shock in the transfected B16 cells,

DISCUSSION
DNA vaccines were broadly utilized in animals; however,they are slightly applied in clinical studies because of their low
immunogenicity. Recently, several approaches have been
evaluated to improve the potency of DNA vaccines such asadvancement in design of the DNA vector, the use of adjuvants
and the delivery systems [14]. The use of liposome as a carrierhas become a popular DNA delivery method in vitro. Plasmid
DNA can be bound to the liposome which facilitates itsdelivery into the cells. In one study, transfection of enhanced
green fluorescent protein (EGFP) using Lipofectamine 3000 has
shown strong expression of EGFP in HEK293 cells, mouseprimary cortical neurons and human umbilical vein endothelial
cells (HUVECs). Also, Cx43 shRNA lentivirus combined with
Lipofectamine 3000 transfection reagent has been shown toachieve ~90% Cx43 knockdown efficacy in HUVECs [15]. In
another study, Zhou et al. have shown that the expression ofsag4 gene cloned in pEGFP-C1 eukaryotic vector in HEK 293T cells, using Lipofectamine 2000 for development of a DNA
vaccine against T. gondii infection [16].
Recombinant DNA technology allows the expression of
antigens in their native conformation and thereby induction ofcellular immunity against pathogens such as HIV-1. Based on
previous studies, HIV-1 Nef has been known as a promising
antigen candidate for HIV-1 vaccine development due toconservation and immunogenicity as well as harboring several
T- and B-cell epitopes [7, 17, 18]. For evaluating the delivery

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5. Liu S, Wang S, Lu S. Using DNA Immunization to Elicit Monoclonal
Antibodies in Mice, Rabbits, and Humans. Human Gene Therapy.
2018;29(9):997-1003. doi:10.1089/hum.2018.077
6. Gary EN, Weiner DB. DNA vaccines: prime time is now. Current
Opinion in Immunology. 2020;65:21-7. doi:10.1016/j.coi.2020.01.006
7. Milani A, Bolhassani A, Shahbazi S, Motevalli F, Sadat SM, Soleymani
S. Small heat shock protein 27: An effective adjuvant for enhancement of
HIV-1 Nef antigen-specific immunity. Immunology Letters. 2017;191:1622. doi:10.1016/j.imlet.2017.09.005
8. Dekaban GA, Dikeakos JD. HIV-I Nef inhibitors: a novel class of HIVspecific immune adjuvants in support of a cure. AIDS Research andTherapy. 2017;14(1):1-4. doi:10.1186/s12981-017-0175-6
9. Bolhassani A, Shahbazi S, Milani A, Nadji SA. Small heat shock proteins
B1 and B6: Which one is the most effective adjuvant in therapeutic HPV
vaccine?. IUBMB life. 2018;70(10):1002-11. doi:10.1002/iub.1892
10. Milani A, Basirnejad M, Shahbazi S, Bolhassani A. HSP roles asbiomarkers and antigens in bacterial and viral infections. Journal of Medical
Microbiology and Infectious Diseases. 2016;4(1):1-7.
11. Milani A, Basirnejad M, Bolhassani A. Heat-shock proteins in diagnosis
and treatment: an overview of different biochemical and immunological
functions. Immunotherapy. 2019;11(3):215-39. doi:10.2217/imt-2018-0105
12. Delneste Y, Larochette V, Jeannin P. Immune Properties of HSP70.
InHSP70 in Human Diseases and Disorders 2018 (pp. 173-203). Springer,
Cham. doi:10.1007/978-3-319-89551-2_9
13. Milani A, Bolhassani A, Heshmati M. Delivery of HIV-1 Nef linked toheat shock protein 27 using a cationic polymer is more effective thancationic lipid in mammalian cells. Bratislavske lekarske listy.
2017;118(6):334-8. doi:10.4149/BLL_2017_064
14. Suschak JJ, Williams JA, Schmaljohn CS. Advancements in DNA
vaccine vectors, non-mechanical delivery methods, and molecular adjuvants
to increase immunogenicity. Human vaccines & immunotherapeutics.
2017;13(12):2837-48. doi:10.1080/21645515.2017.1330236
15. Shi B, Xue M, Wang Y, Wang Y, Li D, Zhao X, et al. An improved
method for increasing the efficiency of gene transfection and transduction.
International journal of physiology, pathophysiology and pharmacology.
2018;10(2):95.
16. Zhou J, Wang L. SAG4 DNA and peptide vaccination provides partial
protection against T. gondii infection in BALB/c mice. Frontiers in
Microbiology. 2017;8:1733. doi:10.3389/fmicb.2017.01733
17. Kadkhodayan S, Jafarzade BS, Sadat SM, Motevalli F, Agi E,
Bolhassani A. Combination of cell penetrating peptides and heterologous
DNA prime/protein boost strategy enhances immune responses against
HIV-1 Nef antigen in BALB/c mouse model. Immunology letters.
2017;188:38-45. doi:10.1016/j.imlet.2017.06.003
18. Rostami B, Irani S, Bolhassani A, Ahangari Cohan R. Expression of the
Recombinant Hsp20-Nef Protein for Evaluation of its Immunogenicity
against HIV-1 Nef using Indirect ELISA. Pathobiology Research.
2019;22(3):135-40.
19. Wang T, Green LA, Gupta SK, Kim C, Wang L, Almodovar S, et al.
Transfer of intracellular HIV Nef to endothelium causes endothelial
dysfunction.
PLoS
One.
2014;9(3):e91063.
doi:10.1371/journal.pone.0091063
20. Yamaoka A, Guan X, Takemoto S, Nishikawa M, Takakura Y.
Development of a novel Hsp70-based DNA vaccine as a multifunctional
antigen delivery system. Journal of controlled release. 2010;142(3):411-5.
doi:10.1016/j.jconrel.2009.11.005
21. Zong J, Wang C, Wang Q, Peng Q, Xu Y, Xie X, Xu X. HSP70 andmodified HPV 16 E7 fusion gene without the addition of a signal peptide
gene sequence as a candidate therapeutic tumor vaccine. Oncology reports.
2013;30(6):3020-6. doi:10.3892/or.2013.2742
22. Andersson HA, Kim YS, O'Neill BE, Shi ZZ, Serda RE. HSP70
promoter-driven activation of gene expression for immunotherapy using
gold nanorods and near infrared light. Vaccines. 2014;2(2):216-27.
doi:10.3390/vaccines2020216
23. Bolhassani A, Shahbazi S, Agi E, Haghighipour N, Hadi A, Asgari F.
Modified DCs and MSCs with HPV E7 antigen and small Hsps: Which one
is the most potent strategy for eradication of tumors?. Molecularimmunology. 2019;108:102-10. doi:10.1016/j.molimm.2019.02.016
24. Deng H, Gao X, Peng H, Wang J, Hou X, Xu H, Yang F. Effect ofliposome mediated HSP27 transfection on collagen synthesis in alveolar
type II epithelial cells. Molecular medicine reports. 2018;17(5):7319-24.
doi:10.3892/mmr.2018.8744

the expression of Hsp70 protein was ~60% compared to < 1%of the transfected cells without the heat-shock [22]. Moreover,heat-shock has led to an increase in the penetration of Hsp27
and HPV16 E7 DNA constructs, and further expression ofproteins in the mesenchymal stem cells. The transfection
efficiencies of pEGFP-E7, pEGFP-Hsp27 and pEGFP-E7 +pEGFP-Hsp27 have been reported to be ~26.5, ~34 and~40.2%, respectively, using Lipofectamine 2000. However,after heating at 42°C for 2 hours, these efficiencies wereincreased to ~51.8, ~60.5 and ~77.5%, respectively [23]. To
investigate the effects of Hsp27 on collagen expression during
TGF-β1-induced differentiation, the Hsp27 plasmid DNA has
been transfected using various Lipofectamine 2000
reagent/DNA ratios in A549 cells in which 5 reagent/DNA
ratios have been assessed, namely 20:8, 25:8, 30:8, 20:9 and20:10. The flow cytometry data then have revealed that thetransfection efficacies were approximately 83, 76, 76, 70, 74%,
respectively. These results have indicated that the 20:8 ratio
was the optimal liposome to plasmid ratio for using in their next
experiments [24].
In current study, the percentage of Hsp27, Hsp27-Nef,
Hap70, Hsp70-Nef and Hsp70 + Nef expression in HEK-293T
at 2:1 ratio (Lipofectamine 2000 reagent/DNA) was 64.22 
0.8, 57.1  0.7, 68.8  1.0, 61.7  0.7 and 77.4  1.5 %,respectively. Herein, the potency of Hsp70 was more than
Hsp27 for increasing Nef-GFP expression in HEK-293T cells.
On the other hand, the delivery of pEGFP-hsp70 along with
pEGFP-nef increased the rate of GFP population in the cells,
indicating a high transfection efficiency as an immunization
strategy for the future attempts. Generally, various factors
including the transfection reagents, the cell types, the degree ofconfluency, the DNA quality and quantity, and thereagent/DNA ratios are important parameters that can greatly
influence the transfection efficiency. In conclusion, ourobservations showed that Hsp70 could significantly deliver Nef
into the cells and increase its expression as compared to Hsp27.
Thus, these constructs will be used as an antigen candidate fordevelopment of a HIV-1 DNA vaccine in an animal model innear future.

ACKNOWLEDGEMENT
The authors would like to thank all those who helped us in thisstudy.

CONFLICT OF INTEREST
The authors report no conflicts of interest.

REFERENCES
1. Alter G, Barouch D. Immune correlate-guided HIV vaccine design. Cell
host & microbe. 2018;24(1):25-33. doi:10.1016/j.chom.2018.06.012
2. Liang B, Li H, Li L, Omange RW, Hai Y, Luo M. Current advances in
HIV vaccine preclinical studies using Macaque models. Vaccine.
2019;37(26):3388-99. doi:10.1016/j.vaccine.2019.04.094
3. Robinson HL. HIV/AIDS Vaccines: 2018. Clinical Pharmacology &Therapeutics. 2018;104(6):1062-73. doi:10.1002/cpt.1208
4. Seddiki N, Lévy Y. Therapeutic HIV-1 vaccine: time for
immunomodulation and combinatorial strategies. Current Opinion in HIV
and AIDS. 2018;13(2):119-27. doi:10.1097/COH.0000000000000444

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The Effects of Heat Shock Proteins on Delivery of HIV-1 Nef Antigen in Mammalian Cells