Bachelor Biomedical Sciences
Thesis
Cloning and expression of eCD4IgG for
lentiviral-based dendritic cell therapy in
HIV-1
Landau Laboratory
New York University, NYC
June 30th 2020
Student: June Vive Verhaak
Supervisor: Nathaniel Landau
Department of Microbiology,
New York University, NYC.
Table of Contents
Abstract ……….………
Introduction ……….………..………
Materials & Methods ………..……….………
A. Method 1: Construction of the amplicon
pLenti.hCD40L.P2A.eCD4IgG through overlap PCR ……….………. B. Method 2: Construction of the amplicon
pLenti.hCD40L.P2A.eCD4IgG
. ……….………..
I. Design of the eCD4IgG construct ………..…. II. Design of the pLenti.hCD40L.Apa1.Sal1 construct ..………..……… III. Blunting of the vector pLenti.puro’ ..………..………. IV. Ligation of pLenti vector and
The BamH1.hCD40L.P2A.apa1.Sal1 insert ……….………
Results.……….………..
A. Method 1: Construction of the amplicon
pLenti.hCD40L.P2A.eCD4IgG through overlap PCR
………
B. Method 2: Construction of the ampliconpLenti.hCD40L.P2A.eCD4IgG.
….………..
I. Design of the eCD4IgG construct ………..…. II. Design of the pLenti.hCD40L.Apa1.Sal1 construct ..………..……… III. Blunting of the vector pLenti.puro’ ..………..………. IV. Ligation of pLenti vector and
The BamH1.hCD40L.P2A.apa1.Sal1 insert ……….………
Discussion .………..……….
Acknowledgments ...………..…………..
References ….………..…………
Supplements ….………..………..
3 3 5 6 7 7 8 8 9 10 10 11 11 11 12 12 14 15 16 16 18Abstract
Research has been conducted on multiple strategies to eradicate human immunodeficiency 1 (HIV-1) from infected individuals. A robust entry inhibitor should bypass the search for a traditional cure. HIV-1 broadly neutralizing antibodies (bNAbs) have been found to neutralize HIV-1 isolates, but 10-50% of HIV-1 isolates are neutralized inefficiently. For the highly variable virus, it is necessary to design a vaccine that elicits a specific antibody that is effective against the most diverse circulating HIV-1 isolates (Diskin et al., 2011, Walker et al., 2011).
Therefore, eCD4Ig is a fusion of CD4IgG with a CCR5 mimetic sulfopeptide. This antibody binds strongly and simultaneously to the two most preserved regions of the HIV-1 envelope (Env). Therefore eCD4IgG is able to neutralize efficiently to a diverse panel of resistant HIV isolates (Gardner et al., 2015). This report develops a lentiviral vector-based dendritic cell vaccine in which the antibody sequence that expresses for eCD4IgG is fused to hCD40L. This paper demonstrates the construction of eCD4IgG through cloning necessary for entry-inhibition of HIV-1 into the host immune cells. Through two different methods of cloning, the researchers attempt to obtain a correct lentiviral vector expressing eCD4IgG and hCD40L which could be a promising tool for a future HIV research.
Introduction
Globally, in 2019 nearly 1.7 million people were newly infected with human immunodeficiency virus type I (HIV-1) (UNAids, n.d). Thus, HIV-1 still remains one of the most public health challenges globally. New concepts have arisen regarding the development of a vaccine for HIV-1. Around 24.5 million HIV-1 infected patients undergo lifelong treatment with antiretroviral therapy, which is recognized as one of the most effective ways to prevent HIV transmission. However, HIV still persists in cells (Chun et al., 2008)
The HIV envelope attaches to two different receptors to deliver the viral payload into immune cells. The HIV-1 envelope’s glycoprotein is composed of a surface gp120 domain. First, gp120 associates with CD4, its cellular receptor, which induces a conformational change in Env. This association allows the envelope protein to change shape and exposes a hidden region that will bind to coreceptor CCR5 on the immune cells. Association with CCR5 will enable the fusion of the viral and the cellular membranes. HIV then injects its genetic material into the cells and infects and kills them by making copies of itself (Wilen et al., 2012). Knowledge of the HIV-1 envelope glycoprotein is necessary for such the design of a vaccine for HIV-1. Broadly Neutralizing antibodies (bNAbs) target most of Env’s coat. The most potent antibodies target the CD4-binding site or the gp120 interface. Some antibodies have been observed to suppress viral rebound during treatment interruption for a small period of time (Scheid et al. 2016; Bar et al, 2016). Previous studies have demonstrated that bNAbs could be deleting transiently activated reservoir cells or be controlling the present infection (Chun et al., 2014). However, despite the positive results from these studies, the bNAbs are not able to neutralize all HIV isolates (Huang et al., 2016; Huang et al., 2012).
Mutations in Env through a high rate of reverse transcriptase make it possible for HIV-1 to escape the antibody treatment. In addition, the bNAbs bind mostly to unconserved regions of Env. To work around this issue, studies have focused on a cocktail of bNAbs that target diverse epitopes instead of a treatment with a single antibody (Wagh et al., 2016; Klein et al., 2012 9). Consequently, the most potent bNAbs inefficiently neutralize more than 10% of HIV-1 isolated from blood tested with 50% inhibitory
concentrations (Huang et al., 2016; Huang et al., 2012 ). Most of the infection-inhibitors bind to epitopes that contact-variable Env residues. This makes it easier for viral escape.
Previous reports have found that the most conserved regions of Env could lead to the eventual design of vaccine of HIV-1 (Rizzuto et al., 1998; Kowalski et al., 1987 ). Those conserved regions are CD4-binding site and its coreceptor binding sites (Huang et al., 2007; Rizzuto et al., 1998). Since four decades, it is of
knowledge that HIV’s receptor on host cells is CD4. (Dalgeish et al., 1984; Klatzmann et al., 1984). The immunoadhesin form of CD4 has its domains integrated to an IgG Fc. This form can inhibit contagion of most HIV-1 isolates, but has a low affinity with gp120 that is lower than those of the bNAbs (Moebius et
al., 1992). Furthermore, CD4-Ig is able to stimulate HIV-1 infection in cells. (Hoxie et al., 2010; Sullivan et al., 1998). Moreover, the sulfopeptides from coreceptor CCR5 have been studied but resulted in
low-affinity binding to Env when CD4 is not present (Farzan et al., 2000; Dorfman et al., 2006). Two targets are better than one, leading to the engineering of the junction of CD4Ig and CCR5 which would increase avidity binding to Env.
Consequently, a novel entry inhibitor (eCD4Ig) was designed that targets the conserved sites of Env (Gardner et al., 2015). eCD4Ig is engineered by separate functional protein domains that fuse into a single novel protein. It is composed of CD4 domains 1 and 2 joined to a feature from the human antibody Fc IgG1 domain. CCR5, which prevents HIV from escaping by binding to gp120, is fused to the C-terminus of CD4Ig (Figure 1). Both the CCR5 mimetic sulfopeptides and the one CD4 arm bind Env which leads to the
suppression of HIV’s entrance into the host cells.
Farzan et al., concluded that at low concentrations, eCD4Ig bound cell surfaces expressed Env trimers more efficiently than CD4Ig. The researchers demonstrated that AAV-delivered rhesus eCD4Ig is less
immunogenic than bNAbs, and after macaques that were injected with the eCD4Ig they were protected from multiple infectious doses. This study demonstrated that eCD4IgG that is expressed via an AAV vector neutralized a larger range of virus of HIV-1 than bNAbs ( Gardner et al., 2015 ).
New strategies for HIV vaccines are in development, one example being the dendritic cell-based vaccine. Patient monocytes are isolated to be differentiated into monocyte-derived dendritic cells in culture. The dendritic cells are then transduced with a lentiviral vector that encodes a certain antibody that is coupled
lentiviral vector-based dendritic cell vaccine with a CD40 ligand (CD40L) that is co-expressed with a HIV-1 antigen. The expression of CD40L will facilitate the binding to its receptor CD40 and the dendritic cells will undergo maturation and proliferation (Mackey et al., 1998). A limitation of previous approaches has been the involvement of eCD4IgG for lentiviral vector- based dendritic cell vaccine.
For this report, researchers performed cloning and tested the expression of eCD4IgG for its ability to be transduced in dendritic cells for lentiviral-based dendritic cell therapy.
The vectors’ package genes encode for hCD40L, P2A and eCD4IgG. The eCD4IgG and hCD40L sequences will be separated by P2A, a self-cleaving peptide. This cleavage allows eCD4IgG to be released as an antibody. The sequence coding for hCD40L, will stimulate a strong response to the antigen by the transduced DC’s. This platform first involved the overlap of two separate fragments into
pLenti.BamH1.hCD40L.P2A.eCD4IgG.Sal1. Due to Covid-19, the researchers were not able to pursue the experiment, so the following experiments were performed by a previous colleague. The resulting amplicon was then cloned with ligation into expression vectors, which were then used directly to transfect 293T cells for protein production. The protein expression was evaluated through western blotting. In the second experiment (performed by the researcher), the amplified eCD4IgG.Apa1.Sal1 was cloned into
pLenti.hCD40L.P2A.BamH1.Apa1.Sal1 through a new method.
Materials and methods
Figure 2. Diagrams of the lentiviral vectors expressing human CD40L (hCD40L) or eCD4IgG. The vectors are
derived from pLenti.CMV.GFP.puro, a promotor HIV-1 based lentiviral vector that has a 5’ cytomegalovirus (CMV) promotor. The vectors express, hCD40L or eCD4IgG. The hCD40L fusion proteins contained a
picornavirus (P2A) self-cleaving sequence (Szymczak et al., 2004).
CMV hCD40L P2A SL9 LTR hCD40L LTR LTR CMV eCD4IgG LTR eCD4IgG LTR CMV hCD40L P2A eCD4IgG LTR hCD40L-eCD4IgG
A. Method 1: Construction of the amplicon pLenti.hCD40L.P2A.eCD4IgG through
overlap PCR
To construct the construct pLenti.hCD40L.P2A.eCD4IgG, an amplicon was constructed that expressed hCD40L fused to the sequence of eCD4IgG microbody.
The first amplicon, pLenti.hCD40L.SL9 (5µL), was amplified with 5µL of forward primer BamH1.hCD40L.RK.f (5uM) and 5µL of reverse primer P2AeCD4IgG.RK.f (5uM). For the second amplicon, the template eCD4IgG-CMVR (5µL) was amplified by 5µL of the forward primer P2A.eCD4IgG (5µL) at the 5’ end and 5µL of the reverse primer eCD4-IgG.Sal1 (5µL) at the 3’ end. To each mix, 1µL of 10mM dNTP’s, 10µL of Phusion buffer 0.5µL of 2U/µL Phusion polymerase and 23.5µL of H20 was added. The PCR was run under certain PCR conditions (Supplement 1) with the SimpliAmp Thermocycler (Applied Biosystems). The PCR
amplification was run at 150V and checked on 0.8% agarose gel by gel electrophoresis.
To remove the enzymes, extra nucleotides, primers and buffer components, the DNA from the PCR was purified with the Wizard clean-up kit Promega.
To construct hCD40L.P2A.eCD4IgG, hCD40L (1µL) was fused to P2A and the sequence coding for eCD4IgG (1µL) by overlapping PCR. The forward primer designed for this reaction was 5µM BamH1.hCD40L (5µL), and 5µM Sal1.eCD4IgG (5µL) was used as the reverse primer. To this mix was added, 26.5µLH20, 0.5µL Phusion polymerase, 10µL of 5X Phusion buffer and 1µL of 10mM dNTP’s. The overlap PCR was proceeded with under certain PCR conditions (Supplement 2). The PCR amplification run at 150V and was checked on 0.8% agarose gel.
The amplicon was cleaved with enzymes BamH1 and Sal1, and it was ligated to similarly cleaved pLenti.puro. The lentiviral vectors stocks for pLenti plasmids were prepared by co-transfection of 293T cells. The 293T cells were plated and added in a 10cm dish. The cells were plated on a 6-well plate and incubated at overnight at 37°C in DMEM. Next, the cells were transfected for western blot. In the first reaction, lipofectomeme 2000 and Optim-mem were added. In the second reaction, the plasmid and the Opti-mem were mixed. Both reactions were combined and overlaid on 293T cells at 37°C for 4 hours. After the media was changed, the cells were incubated for 2 to 3 days at 37°C. Third, the samples were prepared for western blot. After the cells were washed with 1mL PBS, 150-300µL of lysis buffer (including 1/100th of protease inhibitor) was added. After the solution was centrifuged, it was stored at -80°C.
A BCA assay was performed to determine the concentration of eCD4IgG. The cells were removed from the dishes by removing the medium; trypsine was added, and were washed with PBS. A mix of NP40 lysis buffer with 1/100th of protease inhibitor was added. The protein concentrations in lysate were determined using the BCA protein assay reagent. The samples were run on SDS-PAGE gels (NuPAGE Bis-Tris gels from Invitrogen were used), and were run at 150V for 1 hour using prestained protein molecular weight markers. The membrane was activated in methanol for 5 minutes. The gel was taken out, transferred to the membrane and immersed in a transfer buffer. It was then run at 40V for 1 to 2 hours. Afterwards, the researchers added PBS 20mls of blocking solution (TBST milk) in a box with the membrane, and the mixture was incubated and shaken at room temperature for 1 hour. The next addition was the first antibody, which was the human monoclonal anti-CD4, in 3 mls of TBST milk and incubated it for 1 hour. After the antibody was washed off twice in TBST, researchers added the solution containing TBST with the AP-conjugated second antibody, the HRP anti-mouse IgG, and left it for incubation for 1 hour. After the second antibody was washed off with TBST, the researchers developed the blot and imaged it on a Licor machine. This western blot allowed for the detection of the protein eCD4IgG.
B. Method 2: Construction of the amplicon pLenti.hCD40L.P2A.eCD4IgG.
This section involved the testing of another method for the production of the construct pLenti.hCD40L.P2A.eCD4IgG.
I. Design of the eCD4IgG construct
First, the template eCD4IgG.CMVR (5µL) was amplified with the forward primer 5µM
Apa1.eCD4IgG.spacer.f (5µL) and 5µL of the reverse primer eCD4IgG.Sal1 (5µM). In a separate reaction, on the same template a different forward primer was used: 5µL of Apa1.eCD4IgG.nospacer.f (5µM). To both mixes, 1µL of 10mM dNTP’s, 10µL of Phusion buffer, 0.5µL of 2U/µL Phusion polymerase and 23.5µL of H20 were added. Specific PCR conditions were set up (Supplement 3). The PCR amplification was checked on 0.8% agarose gel, and both PCR reactions were purified.
Second, the amplicon (20µL) was cleaved with Apa1 (4µL) and Sal1 (4µL) with help of 10x cut smart buffer (8µL) and H2O (44µL). The reaction was then incubated at 37°C for 1 hour. As the enzyme Sal1 is not as strong as Apa1, 2µL of Sal.1 was pipetted, and the reaction was again incubated at 37°C for 1 hour. The DNA was checked on 0.8% agarose gel by gel electrophoresis at 150V. The bands of the gel were checked
(a) (b)
under UV light, and then they were cut and purified. At that point, the amplicon eCD4IgG.Apa1.Sal1 cleaved at the both ends: at Apa.1 at 5’ end, and Sal1 at the 3’ end.
II.
Design of the pLenti.hCD40L.Apa1.Sal1 construct
The template hCD40L.P2A.SL9 (5µL) was used to amplify hCD40L through PCR using the 5µL of forward primer BamH1.hCD40L.Apa1.RK.f (5µM) and 5µL of the reverse primer hCD40L.P2A.Apa1.Sal1 (5µM). To this mix, 1µL of 10mM dNTP’s, 10µL of Phusion buffer (5X), 2U/µL Phusion polymerase (0.5µL) and 23.5µL of H20 was added. The PCR product was run, under specific conditions (Supplement 4), purified and checked on 0.8% agarose gel at 150V for 15 minutes.
Following the amplification, the insert BamH1.hCD40L.P2A.Apa1.Sal1 (20µL) was cleaved with 4µL of BamH1 (20 000 U/µL) and 4µL of Sal1 (20 000 U/µL) (Supplement 5). The DNA was checked in a 0.8% agarose gel, at 150V for 15 minutes. The gel was scanned under iBright CL1000 from Invitrogen (Thermo Fisher Scientific). The bands of the restriction enzyme digestion of BamH1.hCD40L.P2A.Apa.Sal1 were cut under UV light and were gel purified.
III. Blunting of the vector pLenti.puro’
The pLenti vector of the plasmid has an Apa1 site at 7,348kb. This Apa1 site had to be removed so that it would not interfere with the eCD4IgG insert. Blunting was performed to remove this extra Apa1 site. First, restriction enzyme digestion took place on 1µg/µL of pLenti.GFP plasmid(5µL) with a Cut Smart buffer (8µL), the enzyme (50 000U/mL) Apa1 (2µL) and H2O (65µL). The reaction was visualized on a 0.8% agarose gel at 150V for 15 minutes and was gel purified.
Second, the ends were blunted with 3’ overhang removal and 3’ recessed (5’overhang) end by filling in with T4 DNA polymerase. The DNA was solved in ddH2O (20µL), and supplemented with 100µM dTNPs (0.25µL). Additionally, 1µL of 1 unit of T4 DNA polymerase (3000 U/ML) per microgram DNA was added, and a 10X T4 Polymerase buffer (2.5µL) was used. The reaction was incubated for 10 minutes at 37°C. Ultimately, the DNA was run on a 0.8% agarose gel at 150V, the band was cut under UV light and gel purified.
Third, the blunted ends were ligated. The ligation reaction was composed of 2µL of the vector, 2µL of the DNA from blunting reaction, the enzyme T4 DNA ligase (1µL) and 10X T4 Ligase buffer (2.5µL). Afterwards, the ligase was inactivated by incubating at 37°C for 15 minutes.
Next, restriction enzyme digestion of the ligated product (25µL) was performed with Apa1 (1µL). The reaction contained 10µL of H20 and 4µL of CutSmart Buffer. Before the recombinant plasmid was transformed into E.Coli HB101 competent cells, it underwent a phenol precipitation so that the enzymes and buffer were removed. During transformation, 2µL of plasmid was transformed into 40µL HB101 cells. After electroporation, 1mL SOC media was added to the cuvette, and 150µL was plated on LB agar and ampicillin plates and kept overnight. The colonies obtained from the plate were grown overnight in 2X YT + Ampicillin media at 200 rpm at 37°C overnight. Next day, the plasmid was isolated from the overnight culture.
To test if the Apa1 site was cleaved out properly, researchers performed restriction enzyme digestion on the plasmid that was obtained from the mini preps. To check this, H20 (14µL) and the CutSmart buffer (2µL) were added to the plasmid (2µL), which was cut by the enzymes Apa1 (1µL) and BamH1 (1µL). The reaction was incubated at 37°C for 1 hour. The plasmid was visualized on a 0.8% gel agarose through gel electrophoresis on a thick well at 150V for 15 minutes. The colonies that presented only 1 band were selected, grown and inoculated in large cultures overnight in 300ML of 2X YT Media and Ampicillin at 37°C. The overnight plasmids were isolated through maxi prep plasmid of the Sigma Gel Elute HP plasmid
MaxiPrep kit. After the plasmid was isolated, researchers performed a restriction enzyme digestion on the blunted template (10µL) with 2µL of the enzymes BamH1 (20 000 U/mL) and 2µL of Sal1 (20 000 U/mL) (Supplement 6 ). Ultimately, it was run on a 0.8% agarose gel at 150V. The bands were cut under UV light and were gel purified following the use of the Wizard Gel clean-up system, Promega.
IV. Ligation of pLenti vector and BamH1.hCD40L.P2A.apa1.Sal1 insert
The sequence for BamH1.hCD40L.P2A.Apa1.Sal1 (4µL) was ligated into the vector (2µL) as the Apa1 site was removed with help of the T4 DNA ligase (0.5µL), its 10X T4 Ligase buffer (1µL) and 2.5µL dH2O. The reaction was incubated at 25°C for 1 hour, and the 2µL of the ligated plasmid was transformed into 40µL of HB101 comp cells. The plasmid was plated on pre-warmed LB-Ampicillin plates which were incubated at 37°C overnight.
After the colonies were collected, the plasmid was isolated through mini plasmid preparation. This was followed by a restriction enzyme digestion to check the presence of the insert. In this reaction, BamH1 (1µL), Sal1 (1µL) and 10X NEB Buffer 3.1 (2µL) were combined with 14µL of H2O into the cleavage of the sequence at the specific sites. The digestion reaction was incubated at 37°C for 1 hour and run for 15 minutes on 0.8% gel agarose at 150V. Colonies were picked for maxi prep and the plasmid was sent for sequencing.
Results
1. Method 1: Construction of the amplicon pLenti.hCD40L.P2A.eCD4IgG through
overlap PCR
Both amplicons BamH1.hCD40L.P2A and P2A.eCD4IgG.Sal1 were amplified with PCR to construct the pLenti.hCD40L.P2A.eCD4IgG. First, the gel agarose of the amplification of amplicon
pLenti.BamH1.hCD40L.P2A showed strong bands at the expected size (900bp) (Figure 3a). Second, after PCR amplification for fragment P2A.eCD4IgG.Sal1, the expected bands were similarly strongly observed on the agarose gel (1.4 kb) (Figure 3a). Subsequently, the gel slice of pLenti.BamH1.hCD40L.P2A and
P2A.eCD4IgG.Sal1 containing the interested DNA were excised and purified according to the Wizard Gel clean-up (respectively Figure 3b, 3c). After DNA purification, the amplicons BamH1.hCD40L.P2A and P2A.eCD4IgG were amplified with overlap PCR. The expected size of the bands was 2.3 kb, but no clear bands were present on the gel agarose (Figure 3d). However, the researchers’ previous colleague at the lab, had positive results at the overlap PCR-the reaction worked. Thus, a Western blot was set up to determine the concentration of eCD4IgG.
After the application of SDS-Page to separate the molecules by weight, blocking and detection allowed for the attachment of the primary antibody, the secondary antibody and the enzyme substrate to the protein of interest, eCD4IgG. The gel electrophoresis demonstrated that eCD4IgG was present in the
2. Method 2: Construction of the amplicon pLenti.hCD40L.P2A.eCD4IgG.
I.
Design of the eCD4IgG construct
The template eCD4IgG.CMVR was amplified and the expected size of the DNA was confirmed by gel at 1.4kb (Figure 6a). In parallel, the template was amplified with a different forward primer
Apa.1eCD4IgG.nospacer.f with the expected size at 1.4 kb (Figure 6b). After the template was amplified, both target DNA were purified. Clear bands demonstrated the presence of the purified DNA at 1.4kb (Figure 6c, Figure 6d).
The purified PCR products of eCD4IgG.Apa1.Sal1 were digested with Apa1 and Sal1 restriction enzymes. The DNA were identified and size-separated by gel electrophoresis on 0.8% agarose gels (1.4kb) (Figure 6e). Next, the DNA gel band was excised and purified. Both bands were observed at 1.4kb (Figure 6f).
II.
Design of the pLenti.hCD40L.Apa1.Sal1.construct
The template hCD40L.P2A was amplified and the agarose gel confirmed the presence of the insert at the expected size of 900bp (Figure 7a). After the amplification, the target DNA was purified (Figure 7b). After the genomic DNA samples were cleaved with the restriction enzymes BamH1 and Sal1, a strong band was observed on the gel electrophoresis at 900bp (Figure 7c). The target DNA was excised from the gel and purified (Figure 7d).
III.
Blunting of the vector pLenti.puro’
In parallel, the pLenti.GFP was blunted to remove the Apa1 site at 7,348kb. In the first step, the restriction enzyme Apa1 cut the vector at the Apa1 site. The DNA was analyzed by gel electrophoresis, and 1 band was expected at 8.6kb (Figure 8a). The gel slice containing the digested DNA was purified and analyzed by gel electrophoresis. Researchers observed a difference in brightness in each well in the agarose gel of the pLenti.GFP, potentially because the amount inserted in the gel was different for each sample (Figure 8b). Next, the DNA was blunted and the agarose gel showed a clear intense band at 8.6kb (Figure 8c). This band was cut and gel purified (Figure 8d). A weak band was observed on the agarose gel, which could be
After the recombinant plasmid was ligated, it was transferred into E.Coli HB101 competent cells. Twelve colonies were picked and grown overnight, their plasmid was isolated and the DNA was digested with restriction enzymes Apa1 and BamH1. The DNA was analyzed by gel electrophoresis, and one band was expected representing the vector at size 8.6kb. If a second band would be present, not to be expected, it would represent the Apa1 site, with size 2.5kb (Figure 9a). The samples with one band were selected and grown overnight. Next day, a maxi plasmid prep was conducted, and the concentration of DNA was calculated through Nanodrop (Table 1 in Supplement 7).
As the DNA yield was sufficient, the plasmid was sent for sequencing at Genewiz. The sequencing results confirmed the absence of the Apa1 site (Supplement 8). In parallel, the vector was digested by the
restriction enzymes BamH1 and Sal1. The gel agarose showed the presence of the lower band GFP (850bp), and the vector that was clearly present (8.6 kb) (Figure 10).
IV.
Ligation of pLenti vector and BamH1.hCD40L.P2A.apa1.Sal1 insert
Next, the insert BamH1.hCD40L.P2A.Apa1.Sal1 was ligated into the pLenti vector. Then, the recombinant plasmid was transferred into E.Coli HB101 competent cells. The next day, the colonies from the different plates were selected.
Isolation of the plasmid was performed on the picked colonies, which was followed with restriction digestion with the enzymes BamH1 and Sal1, which could confirm the presence of the insert in the vector. Researchers expected to see two bands on the gel agarose. Most of the colonies presented 2 bands at 8.6 kb (representing the vector) and a band of size 850bp (representing the insert) (Figure 9). The plasmid was sent for sequencing at Genewiz, and showed the presence of GFP instead of the expected insert
Discussion
This report describes two detailed methods for the construction of the pLenti.hCD40L.P2A.eCD4IgG. In the first instance, researchers correctly amplified two fragments using pLenti.hCD40L.SL9 and eCD4IgG-CMVR plasmids. This resulted in the clear presence of bands that identified the available amplicons at the expected sizes. Next, researchers performed an overlap PCR with the amplified fragments. The results of the gel agarose by gel electrophoresis indicated no clear bands of the amplicon hCD40L.P2A.eCD4IgG at the expected size (2.3 kb), resulting in an erroneous cloning. This means that the cloning did not work, and researchers were unable to proceed to the transfection of the 293T cells and measure the protein
expression.
There were multiple potential reasons for the failure of the overlap PCR. First, the conditions for the PCR may not have been adequate for this reaction. Second, different annealing temperatures for the overlap PCR could have been used. Third, another strategy that could have been used for this protocol was starting the reaction without the primers and adding them only after the denaturation step. Another reason for the negative results of this overlap PCR could have been the contamination of the DNA, which could have hindered the joining of the two fragments.
However, the researchers’ previous colleague at the lab, had positive results at the overlap PCR-the
reaction worked. Thus, the resulting amplicon was cloned with ligation into expression vectors, which were then used directly to transfect 293T cells. The protein expression of eCD4IgG was evaluated through
western blotting. The analysis of the western blotting demonstrated the presence of the band at 45 kDa. This indicated the presence of the protein eCD4IgG.
As the first experiment for cloning, concerning the overlap PCR did not produce the expected results, the researchers chose to use a different approach for the production of the construct
pLenti.hCD40L.P2A.eCD4IgG . First, researchers amplified both fragments BamH1.hCD40L.P2A.Apa1.Sal1 and Apa1.eCD4IgG.Sal1 by PCR. Both amplifications occurred correctly because the expected bands were present on the gel agarose at the expected size,- respectively at 900 bp and 1.4kb. Second, the vector plasmid had to be blunted to remove the Apa1 site at 7,348 kb. It was removed correctly as the sequencing results demonstrated that the vector did not contain the Apa1 site. Third, the insert
BamH1.hCD40L.P2A.Apa1.Sal1 was ligated into the blunted pLenti vector. The recombinant plasmid was sequenced which indicated that the band that was expected as the insert BamH1.hCD40L.P2A.Apa1.Sal1 was GFP. This outcome suggested that the cloning of the insert into the vector did not ligate correctly. The ligation could have failed for several reasons. First, the DNA could be damaged because the researchers used UV light for gel purification which might have blocked successful ligation of the insert into the vector. Second, the vector could have had a high background, meaning an incomplete digestion. Third, the time of incubation was just merely 1 hour, which could have had a negative low effect for ligation time. A change for this protocol could be an increase of duration in ligation time.
Two key questions are whether eCD4IgG can be cloned correctly and has a potential protein expression after transducing with 293T cells for lentiviral-based dendritic cell therapy.
This data that was obtained prior to this researcher’s arrival demonstrates that the protein eCD4IgG was expressed correctly in the transduced 293T cells. The lentiviral vectors when encoded with eCD4IgG and co-expressed hCD40L could stimulate the DC maturation and activation. As previous study of Norton et al., 2019 showed, hCD40L when expressed is able to induce DC proliferation. Other previous studies have demonstrated, the expression of eCD4IgG antibody prevents HIV-1 from entering the host cells (Gardner et
al., 2015). Results of this report show that eCD4IgG was expressed correctly, it could function as
entry-inhibitor of HIV-1 into host cells. Thus, it is possible to construct the antibody like eCD4IgG and it could provide a new method for lentiviral dendritic cell based therapy. Other studies (Fetzer et al., 2018) have indicated the breadth of the neutralization of eCD4Ig, depending on its variant. This report focuses on one
variant. As a further test, multiple eCD4Ig variants could be studied if that would support a better
development of potential therapeutic vaccinations for the HIV-1 of infected patients. The clearest benefit of eCD4IgG over bNAbs is its efficiency and breadth, which come from the conserved sites to which
eCD4IgG binds to. Moreover, the dendritic cells could be transfected with this construct. The cells could be injected in humanized mice to test the ability of the transduced dendritic cells to express the antibody eCD4IgG. The suppression of the viral load induced by eCD4IgG could be measured.
Although challenges remain, these observations suggest that this gene therapy that expresses eCD4IgG could provide effective entry inhibition to HIV-1 into host cells.
Acknowledgments
The internship was supported by the Department of Microbiology at the New York University (NYC).
I would like to thank Dr. Nathaniel Landau for giving me the opportunity to do the internship at the Landau Lab, NYU. I would like to thank Ramanjit Kaur (Ph.D.) for the daily guidance and for teaching me the cloning techniques.
I would like to tank Aisha for the use of her data-set and set of experiments that I was not able to finish given the circumstances (Covid-19). I would also like to thank the other members of this lab, Rebecca, Takuya and Luis for helping me and giving me advice on the techniques and for the welcoming atmosphere in the lab.
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Supplements
Supplement 1
The PCR conditions for the amplification of the two individual fragments: plenti.hCD40L.SL9 and eCD4IgG.CMVR
For the initial denaturation, the temperature was set up at 98°C for 30seconds. It was followed by
denaturation, where the reactions were subject to 98°C for 10 seconds. In the next step of annealing, the temperature was lowered to 50°C for 30 seconds allowing for annealing of the primers. The temperature for the elongation step was 72°C for 2 minutes. Multiple cycles are needed for the amplification of the target DNA for millions of copies, so the number of cycles was set at 30. For the final step, the final
extension, the temperature was held at 72°C for 5 minutes. At the end, the PCR products were briefly stored in the reaction chamber at 4°C.
Supplement 2
The PCR conditions for the overlap PCR of pLenti.hCD40L.SL9 and eCD4IgG.CMVR into one amplicon: hCD40L.P2A.eCD4IgG
The reaction chamber was heated to 98°C for 30 seconds during initial denaturation. The following step, the denaturation, involved exposing the reaction to 72°C for 10 seconds at 30 cycles. For the annealing of the primers to the template, two different temperature were used. The first 5 cycles, involved 40°C for 30 seconds. The remaining 25 cycles, involved 50°C for 30 seconds. During the elongation, when the DNA polymerase synthesizes a new DNA strand complementary to the template, the temperature set up was at 72°C for 2 minutes and 30 seconds for 30 cycles. During the final extension, the reaction chamber was set at 72°C for 5 minutes and then left on hold for an indefinite period at 4°C.
Supplement 3
The PCR conditions for the amplification of eCD4IgG (method 2) were as follows.
seconds (for the no.spacer forward primer, the temperature was 55°C, 60°C for 30 seconds). The extension was performed at 72°C for 2 minutes, followed by a final extension at 72°C for 5 minutes. The reaction chamber cooled down at 4°C on hold for an indefinite time period. The denaturation, annealing and extension were performed for 30 cycles.
Supplement 4
The PCR conditions for the amplification of hCD40L
For the initial denaturation the temperature was set up at 98°C for 30 seconds, followed by denaturation at 98°C for 10 seconds. The primers were annealed at 50°C for 30 seconds, and the strands of DNA were extended at 72°C for 2 minutes. These strands were extended for a final step for 5 minutes at 72°C. The reaction chamber cooled down to 4°C for an indefinite time period. The denaturation, annealing and extension were performed for 30 cycles.
Supplement 5
Restriction enzyme digestion of insert BamH1.hCD40L.P2A.Apa1.Sal1 with Bam and Sal1.
For this reaction, the buffer used was the NEB buffer 3.1 (8µL). H20 (44µL) was added, and the enzymes 20 000 U/µL BamH1 (4µL) and 20 000 U/µL Sal1 (4µL), both of which were going to cut on two sites on the template (20µL). The reaction was first incubated for 1 hour at 37°C, then another portion of Sal1 (2µL ) was added and incubated again for another hour.
Supplement 6
Restriction enzyme digestion of the vector pLenti with BamH1 and Sal1
The reaction was composed of 10µL of the template Apa1.Blunted.plasmid, where 2µL of BamH1 and 2µL of Sal1 cleaved the sequence at the specific sites. The extra components added were 58µL of H2O and 8µL of NEB Buffer 3.1.It was incubated for 1 hour at 37°C
Supplement 7
Table 1. DNA concentrations
Yield (µg)
Sample 1 412,78 µg of DNA per 200 µL TE
Supplement 8
The sequencing results show the absence of Apa1 around 7300bp.
