Blocking HIV-1 Infection by Chromosomal Integrative Expression of Human CD4 on the Surface of Lactobacillus acidophilus ATCC 4356

Bacterial strains, plasmids, and primers.The bacterial strains, plasmids, and primers used in this study are listed in Table 2. Escherichia coli strains were aerobically grown in LB broth at 37°C in a rotary shaker. Lactobacillus strains were grown in 5% CO₂ in MRS (Oxoid) broth at 37°C without shaking. Solid medium was prepared by adding 1.5% (wt/vol) agar to the broth. The plasmid constructions were first established in E. coli cells and then transformed into Lactobacillus strains. The antibiotics used for E. coli were 100 µg/ml ampicillin, 100 µg/ml kanamycin, or 150 µg/ml erythromycin, while that used for Lactobacillus bacteria was 5 µg/ml erythromycin.

Construction of the CD4 surface display plasmid.The pTRKH3-ldhGFP vector (19) was used as the backbone for the construction of the recombinant vector. To assemble the CD4 expression cassette, the constitutive ldhL promoter from L. acidophilus ATCC 4356 and green fluorescence protein (GFP) from vector pTRKH3-ldhGFP and the signal peptide sequence of the YSIRK gene (GenBank accession number CBL49691) from L. crispatus ST1 genomic DNA (22) were individually amplified and then fused to a single DNA fragment with SalI and SacI sites by overlapping PCR. Human CD4 domain 1 and 2, with codons optimized for expression in Lactobacillus bacteria, were amplified with SacI and XhoI sites. The short anchor (117 amino acid [aa]) or the long anchor (244 aa) of the serine proteinase (PrtP) from L. casei was amplified from vector pLP401T (21, 32, 33) or synthesized as a whole length sequence by Eurofins with flanked XhoI and PstI sites, respectively. The transcription terminator (Tcbh) of the L. plantarum-conjugated bile acid hydrolase gene was amplified with flanked SalI-PstI and BglII sites. All these fragments were assembled and finally cloned into the SalI and BamHI sites in pTRKH3-ldhGFP to give CD4 anchor vector pWZ486, as depicted in Fig. 1A.

Electroporation.Plasmids were transformed into the Lactobacillus bacteria by electroporation, as described by Majidzadeh Heravi (34) with minor modifications. Briefly, overnight cultures of Lactobacillus cells were diluted (1:50) into fresh MRS medium with 1% glycine and incubated at 37°C without shaking for 2 h. Cells were harvested and treated with 50 mM EDTA (pH 8.0) for 15 min, followed by two washes with ice-cold electroporation buffer (0.5 M sucrose), and resuspended in electroporation buffer (1/100 volume of the initial culture). A total of 50 µl of cells was mixed with plasmid DNA and incubated on ice for 15 min. The mixture was added to an ice-cold 0.2-cm GenePulser (Bio-Rad) cuvette and pulse was immediately applied at the conditions of 10 KV/cm, 200 Ω, and 25 µF. Cells were suspended in 1 ml MRS broth with 2 mM CaCl₂ and 20 mM MgCl₂ and then incubated at 37°C for 4 h. Cells were pooled on MRS plates with 5 µg/ml erythromycin and cultured for 2 to 3 days with 5% CO₂. Clones were picked and grown in MRS medium with erythromycin. A total of 50 µl of cells was collected, washed once with 1 ml PBS, suspended in 50 µl of PBS, and then boiled for 10 min and subjected to PCR verification.

DNA manipulation.DNA manipulations were performed as previously described (35). The restriction enzymes and T4 DNA ligase, calf intestinal alkaline phosphatase (New England Biolabs, MA, USA) and the high-fidelity DNA polymerase (TaKaRa, Japan) were used according to the manufacturer’s instructions. Amplified PCR products were separated on 1% agarose gels and purified using the QIAquick gel extraction kit (Qiagen). Plasmid DNA was purified from E. coli using the Qiagen mini/maxi kit (Qiagen). Genomic DNA from Lactobacillus bacteria was extracted using the GenElute bacterial genomic DNA kit (Sigma). All DNA constructs were verified by DNA sequencing (Eurofins).

Expression of CD4 in E. coli.For fusion expression of GFP-CD4 in E. coli, the fusion DNA fragments were amplified by overlapping PCR with primer pairs 118 + 120 and 119 + 057 (Table 2), then digested with SacI and XhoI, and cloned into the expression vector pET28a (Invitrogen), resulting in pWZ427. Clones of the expression host E. coli BL21(DE3) containing pWZ427 were cultured to optical density at 600 nm (OD₆₀₀) around 0.6 to 0.8 and then induced by 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) for 3 h at 37°C. After induction, the cells were collected by centrifugation, boiled in SDS sample buffer, and subjected to SDS-PAGE and Western blot analysis.

Chromosomal integration of Lactobacillus bacteria.For the chromosomal integration, the E. coli cloning vector pUC18, which is not able to replicate in Lactobacillus bacteria, was used as the basis for the construction of the integration vectors for CD4 expression.

First, the erythromycin resistance gene (erm) from vector pLP401T was chosen as the selective marker of the integrated bacteria. To eliminate the internal SacI site to facilitate subsequent cloning procedures, the gene sequence was amplified through overlapping PCR with primer pairs 001 + 072 and 002 + 073, in which SacI site was mutated. The amplified fragment was digested with EcoRI and cloned into pUC18; the insertion direction was verified by restriction analysis to produce the vector pWZ528.

Second, the sequence in the middle of two reverse transcript genes in the L. acidophilus chromosome was chosen as the integrative site to achieve the lowest adverse effects on the host strain characteristics. Two chromosomal fragments as the homologous upper and down recombination sites were amplified with primer pairs 184 + 185 or 186 + 187, then digested with EcoRI or HindIII, respectively, and inserted into vector pWZ528 consequently. The insertion direction of the two fragments in the final vector pWZ531 was verified by PCR to be the same as the original chromosomal direction. To facilitate the exchange of the upper and down integration sites to other location and even other stains, two rare restriction sites, namely, PacI and PmeI, were inserted flanking the upper and down fragments, respectively.

Third, the entire CD4 expression cassette was digested out from vector pWZ486 with SalI and PstI and subcloned into the same sites of the vector pWZ531, giving the CD4 integrative vector pWZ535.

Western blot analysis.Western blotting samples were run on a 10% SDS-polyacrylamide gel and stained with Coomassie blue to verify protein expression. For the detection of CD4 expression, the SDS-PAGE gel was further transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA) and the membrane was blocked with blocking buffer (5% skim milk in PBS) for 1 h at room temperature and reacted with primary antibody (2000-fold-diluted rabbit anti-CD4 polyclonal antibody; Santa Cruz) overnight at 4°C. The membrane was washed 3 times with wash buffer (0.1% Tween 20 in PBS) and incubated with a secondary antibody (5000-fold-diluted goat anti-rabbit IgG antibody conjugated with horseradish peroxidase [HRP]) for 1 h at room temperature. After washing 3 times with wash buffer, the membrane was developed with ECL substrate (Fisher). The GFP expression was detected with HRP-conjugated rabbit anti-GFP antibody (Santa Cruz) with the similar treatment of the membrane as described above.

Flow cytometry and indirect immunofluorescence microscopy of the CD4-expressing L. acidophilus cells.Cells that reached a density of approximately 5 × 10⁶ cells were collected by centrifugation. Cells were washed twice with 500 µl PBS and suspended in 100 µl PBS with 2 µl anti-CD4 polyclonal antibody (Santa Cruz). After incubation on ice with gentle shaking for 1 to 2 h, the bacteria were centrifuged at 7,000 × g for 3 min at 4°C and washed three times with 500 µl PBS. The cells were subsequently resuspended in 100 µl PBS with 2 µl of goat anti-rabbit IgG conjugated with APC allophycocyanin (APC; Santa Cruz) and incubated on ice with gentle shaking for 1 h. After collecting the bacteria by centrifugation at 7,000 × g for 3 min at 4°C and washing three times with 500 µl PBS, staining was analyzed by flow cytometry using fluorescence-activated cell sorter (FACS) Calibur (Becton, Dickinson), as described previously (36, 37). Images were collected using a Leica TCS-NT/SP confocal microscope (Leica Microsystems, Mannheim, Germany).

Generation of pseudotyped viruses.The 293T cells were plated at a density of 2.0 × 10⁶ cells in a 10-cm plate. Twenty-four hours later, the cells were transfected with an HIV-1 backbone plasmid, pSG3^ΔEnv (NIH AIDS Reagent Program), and an Env expression plasmid using polyethyleneimine (PEI). One day following transfection, additional medium was added to the plate. Three days posttransfection, the supernatants were harvested and, after a short spin to remove cell debris, stored at −80°C. The viral titers were determined by reverse transcriptase assay.

Reverse transcriptase assay.In duplicate, 500 µl of pseudovirus-containing supernatant was spun at 14,000 × g for 2 h at 4°C. Following the spin, the supernatant was removed and the viral pellet was resuspended in a Triton X-100-based suspension buffer and vortexed, followed by three rapid freeze-thaw cycles to lyse the virus. A total of 50 µl of reaction mix [oligo(dT) poly-A and 3H-dTTP; PerkinElmer] was added, and the samples were incubated at 37°C for 1 h in a heating block. Following incubation, the samples were pipetted onto cut squares of DEAE filtermat paper (PerkinElmer), followed by three 10-minute washes in 2× SSC (0.3 M NaCl plus 0.03 M sodium citrate) buffer, and one 10-second wash in 100% ethanol. The filters were dried at room temperature and then analyzed using a scintillation counter to quantify the incorporation of ³H-dTTP into cDNA. The average cpm values from each duplicate were then used to normalize the amount of virus-containing supernatant that was used in subsequent single-round viral entry assays.

HIV absorption.Pseudotyped single-round HIV-1, VSV, and AMLV viruses made from 293T cells were used for the experiment, and the virus titers were measured by reverse transcriptase (RT) activity. The engineered or wild-type bacteria (∼5 × 10⁷/ml) were mixed with the viruses (virus stocks, ∼200 kcpm/ml) in a 1.5-ml microcentrifuge tube. The bacterium and virus mixtures were incubated for 1 h at 37°C with rocking. In some experiments, either soluble CD4 (sCD4) (50 µg/ml) (NIH AIDS Reagent Program) or the QS4120 anti-CD4 monoclonal antibody (30 µg/ml) (EMD Millipore) was added to the bacterium-virus mixture. Then, the tubes were spun for 1 min at full speed to remove the bacteria. The supernatants were collected and the viral titers were determined by RT.

MTT cytotoxicity assay.The MTT assay was performed to determine the cytotoxicity of Lactobacillus cell cultures for the TZM-bl cells used in the neutralization assays. TZM-bl cells were seeded in a 96-well plate at 6,000 cells/well and incubated overnight. The cells were washed with PBS, and then 50 µl of Lactobacillus-cultured Dulbecco’s modified Eagle medium (DMEM) was added to the cells. The Lactobacillus-cultured DMEM was prepared with wild-type L. acidophilus and L. acidophilus-huCD4. Bacteria were incubated in DMEM at 37°C for 1 h. The bacteria were pelleted and the supernatants were applied to the TZM-bl cells. After an overnight incubation, 150 μl of fresh DMEM was added to the cells. Twenty-four hours later, the medium was removed and the cells were washed once with PBS. A 5-mg/ml solution of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2 H-tetrazolium bromide (Sigma-Aldrich) was added to each well. Plates were incubated at 37°C for 4 h. The precipitate was dissolved in 100 μl DMSO and absorbance was measured on an enzyme-linked immunosorbent assay (ELISA) plate reader at 570 nm.

HIV-1 neutralization analysis.Pseudotyped HIV-1 viruses for single-round infection were used in the engineered bacterial neutralization assay. TZM-bl cells which express CD4, CCR5, and CXCR4 were used for the target cells, which allow viral infectivity to be evaluated by measuring the luciferase activity (38). The TZM-bl cells were set at a density of 8.0 × 10³ per well in a 96-well plate. The control bacteria (L. acidophilus cells only) or CD4-expressing integrants were cultured to early log phase (OD₆₀₀, 0.1 to 0.2), and 5 × 10⁷ cells (approximately 5 × 10⁸ cells per OD₆₀₀) were collected. Cells were washed and resuspended in DMEM. The incubation of mixed bacteria and viruses was prepared in a tube for triplicates of each well with 2,500 RT units of viral stock in a final 100-µl infection volume. The tubes with mixed bacteria and viruses were incubated with rocking for 60 min at 37°C. After centrifugation at 10,000 rpm for 3 min to spin down the bacteria, supernatants were transferred to new Eppendorf tubes and loaded into the wells of TZM-bl cells for infection at a final volume of 100 μl. Three days postinfection, the supernatants were removed, the cells were washed once with PBS, and the cells were lysed in 1× passive lysis buffer and frozen at −80°C. The plates were then thawed, and luciferase activity was measured using the Veritas luminometer and beetle luciferin substrate (Promega).

LiCl treatment.To verify whether the expressed CD4 is covalently anchored on the cell wall, cells (5 × 10⁷) were incubated with 100 µl of 5 M LiCl for 15 min and then cells were washed with PBS and resuspended in SDS sample buffer for Western blot analysis. The cell samples used in Fig. 2E were subjected to incubation with a 5 M LiCl solution for 15 min at 37°C. Cells were then washed with PBS, boiled in SDS sample buffer, loaded for SDS-PAGE and Western blot. There was no significant difference between treated and untreated samples, indicating that CD4 was covalently linked to the cell wall through peptidoglycan (39).

Generation of BLT humanized mice.Bone marrow, liver, and thymus (BLT) humanized mice were generated by implantation of human fetal thymic grafts and adoptive transfer of autologous hematopoietic stem cells (CD34) into NOD/SCID/IL2Rγ^−/− (NSG) mice, as described previously (40). For evaluation, flow cytometry was used to detect hCD45, hCD4, and hCD8 cells. Prior to HIV-1 challenge, the BLT mice were treated with 2 mg of medroxyprogesterone subcutaneously to synchronize the estrus cycle of the female mice, allowing their vaginal epithelium to be at a comparable thickness at the time of HIV-1 challenge (41). Animal work was approved by the Institutional Animal Care and Use Committee (IACUC) of the Massachusetts General Hospital.

Bacterial inoculation and HIV-1 challenge of BLT humanized mice.A total of 32 BLT humanized mice, half males and half females, were used for this experiment. Four groups (8 mice per group) were divided into the rectal challenge route group (male mice) and vaginal challenge groups (female mice) as follows: (i) rectal control group, to receive bacterial vector only (RC); (ii) rectal treatment group, to receive hCD4⁺ bacteria (RT); (iii) vaginal control group, to receive bacterial vector only (VC); and (iv) vaginal treatment group, to receive hCD4⁺ bacteria (VT). One mouse (241287) was found dead prior to the start of the experiment and was therefore removed from the RC group.

Bacterial administration was done by direct atraumatic application of 20 µl of the bacterial samples (10¹⁰/ml, frozen samples) into the rectum or vagina. Two hours after bacterial inoculation, the viral challenge followed. All mice were anesthetized with isoflurane inhalation during the experimental process, and their body was kept in an inverted position for 4 min after the bacterial and viral inoculation. In addition, in order to decrease feces/urine during the experiment, all mouse feeding was stopped for 2 h before and after the bacterial and viral inoculation. Note that for vaginal groups of mice, in order to synchronize/prolong the estrous phase for bacterial inoculation, they were injected subcutaneously with progesterone (2 mg/mouse; Depo-Provera, Pharmacia & Upjohn Diagnostics) in a 100-µl volume 5 days before the bacterial inoculation. Viral rectal or vaginal challenges matched the route of bacterial inoculations. HIV-1 JR-CSF (10⁵ 50% tissue culture infective dose [TCID₅₀]) in 10 µl PBS was directly applied intravaginally or rectally using a pipette. Blood samples for viral load detection were collected at day 0 before bacterial inoculation/viral challenge and days 14 and 28 postchallenge.

Statistical analyses.Statistical analyses were performed using GraphPad Prism software (version 6.0). To analyze the engineered bacterial neutralizing activities against HIV-1 viruses, the statistical significances were determined by using the Holm-Sidak t test method with alpha of 5.000%. For humanized mouse data, the statistical significances were determined by using the unpaired one-tailed t test at P value of <0.05.