Yonsei Med J.  2011 Jan;52(1):173-180. 10.3349/ymj.2011.52.1.173.

Efficiency of Recombinant Bacille Calmette-Guerin in Inducing Humoral and Cell Mediated Immunities against Human Immunodeficiency Virus Type 1 Third Variable Domain in Immunized Mice

• Kim YJ

• KMID: 1106453
• DOI: http://doi.org/10.3349/ymj.2011.52.1.173

Abstract

PURPOSE
The third variable (V3) loop of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein has been intensively studied for AIDS vaccine development. Bacille Calmette-Guerin (BCG) is widely used to immunize against tuberculosis and has many advantages as a vaccine vehicle, such as low toxicity, adjuvant potential, low cost, and long-lasting immune-inducing capacity. This work was initiated to investigate the immunogenicity of recombinant BCG (rBCG-mV3) designed to express trimeric HIV-1 V3 loop (mV3) in rBCG-mV3-immunized animals.
MATERIALS AND METHODS
HIV-1 V3-concatamer was cloned into pMV261, a BCG-expression vector, and then rBCG-mV3 was constructed by introducing the recombinant plasmid (pMV-V3). The recombinant BCG was examined with regard to its expression of V3-concatamer and the genetic stability in vivo and in vitro. The immune responses induced by recombinant BCG were tested in immunized mice and guinea pigs.
RESULTS
The rBCG-mV3 expressed detectable amounts of V3-concatamer when induced by single heat-shock. The recombinant BCG was genetically stable and maintained the introduced mV3 gene for several weeks. V3-specific antibodies were clearly detected 6 weeks after inoculation. The antibody titer rapidly increased after immunization up to 10 weeks, and then maintained for over 4 weeks. IgG2a was prevalent in the V3-specific antiserum. The recombinant BCG was also effective in inducing delayed-type hypersensitivity responses in the immunized guinea pigs. rBCG-immunized mice retained substantial amounts of V3-specific T cells in the spleen, even 5 months after the first immunization.
CONCLUSION
Recombinant BCG-mV3 is very efficient in inducing humoral and long-lasting cell-mediated immunity against HIV-1 V3 in the immunized animals.

Keyword

HIV-1 vaccine; V3 domain; recombinant BCG; immunized animal

MeSH Terms

AIDS Vaccines/genetics/*immunology
Animals
BCG Vaccine/genetics/*immunology
Female
Guinea Pigs
HIV-1/*immunology
Humans
Immunity, Cellular/genetics/*immunology
Immunity, Humoral/genetics/*immunology
Mice
Mice, Inbred BALB C
env Gene Products, Human Immunodeficiency Virus/genetics/*immunology

Figure

• 

  Fig. 1 Schematic illustration of experimental procedure. Phsp60, promoter for heat shock protein; MCS, multiple cloning site; aph, gene conferring kanamycin resistance as a selective marker; oriE, E. coli origin of replication; oriM, mycobacterial origin of replication; HIV, human immunodefi-ciency virus; V3, third variable.

• 

  Fig. 2 (A) V3-trimer was cloned into pRSET-B vector, and expressed in E. coli BL21 (DE3). Whole cell lysates or purified protein was separated on a SDA-PAGE. Lane 1, pRSET/B control vector-transformed cell lysate; lane 2, pRSET-mV3-transformed cell lysate; lane 3, Ni+-NTA resin-purified recombinant mV3 protein. (B) Western blot analysis of V3-concatamer (three V3) expressed in bacteria with 2 different antisera. pRSET/B control vector-transformed cell lysate (1) and pRSET-mV3-transformed cell lysate (2) were separated on a SDS-PAGE, and then assessed by Western blot analysis with mouse anti-3V3-antibody (a) and rabbit anti-gp120 antibody (b). SDA-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; V3, third variable.

• 

  Fig. 3 (A) Western blot analysis of rBCG-mV3 lysates with anti-V3 antiserum. Cultures of rBCG-mV3 were heat-induced at 45 for 2 hours in the log phase, and the same amounts of cells were harvested from the culture in 2 week intervals. M, marker; lane 1, BCG-pMV control; lane 2, BCG-mV3 (2 weeks); lane 3, BCG-mV3 (4 weeks); lane 4, BCG-mV3 (6 week) after a single heat-shock; lane 5, mV3-expressing E. coli lysate as a positive control. (B) PCR amplification of the plasmid extracted from the kanamycin-resistant rBCG that was isolated from the spleen of immunized mice was performed with V3-specific primer set. Each lane shows a pMV-mV3 positive control (1), pMV261 negative control (2), and plasmid recovered from the rBCG-mV3-immunized mice 4 weeks (3) and 8 weeks after immunization (4). (C) Western blot analysis of rBCG-mV3 recovered from immunized mice with anti-mV3 sera. Each lane shows BCG-pMV (1), rBCG-mV3 (2), and pRSET-mV3 / E. coli BL21 (DE3) lysate (3). BCG, Bacille Calmette-Guérin; PCR, polymerase chain reaction.

• 

  Fig. 4 rBCG-mV3 induces V3-specific antibody in BALB/c mice. (A) Six-week old BALB/c mice were inoculated with control (transformed with pMV261 empty vector) and recombinant BCG (rBCG-mV3) at a concentration of 1 × 107 cells/mouse intraperitoneally. The titer of anti-V3 antibody in the serum obtained from the immunized mice every 2 weeks were assessed by ELISA as described in the Materials and Methods. Anti-serum was collected from the rBCG-mV3-immunized mice 6 weeks after primary inoculation. (B) Cell lysates or purified mV3 were examined by Western blot analysis with the antiserum obtained from the mice immunized with rBCG-mV3. The arrow indicates the band of purified mV3 protein. (C) C8166 human T cell line was infected with 1 MOI of HIV-1 (HXB2), which was pre-incubated with mouse anti-rBCG-mV3 sera or control anti-BCG sera at 37 for 1 hr. Cells were harvested and lyzed at the indicated time point and then p24 antigen was assessed by Western blot analysis with anti-p24 monoclonal antibody. OD, optical density; PI, postinoculation; MOI, multiplicities of infection; BCG, Bacille Calmette-Guérin.

• 

  Fig. 5 (A) Lymphocyte proliferation assay in response to recombinant-mV3 antigen stimulation in T cell-enriched splenocytes of immunized mice. Spleens were removed from 3 mice per group 10 weeks and 5 months after immunization with rBCG-mV3 and control BCG (BCG-PMV), respectively. Lymphocyte proliferation assay was performed by measuring the [3H]-thymidine incorporation, and is expressed as the mean SD stimulation indices in triplicate and three independent experiments. (B) Anti-mV3 antibody isotyping was performed using a monoclonal antibody-based mouse Ig isotyping kit and expressed by percentage for each isotype and subclass. Anti-serum was obtained from mice 10 weeks after immunization with rBCG-mV3. Anti-serum was pre-cleared with BCG lysates and then used for ELISA. PI, postinoculation; BCG, Bacille Calmette-Guérin.

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