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

Affiliations
  • 1Department of Laboratory Medicine, Sungkyunkwan University School of Medicine, Masan Samsung Hospital, Masan, Korea. kyj7514@unitel.co.kr

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.


Reference

1. Javaherian K, Langlois AJ, LaRosa GJ, Profy AT, Bolognesi DP, Herlihy WC, et al. Broadly neutralizing antibodies elicited by the hypervariable neutralizing determinant of HIV-1. Science. 1990. 250:1590–1593.
Article
2. Choe H, Farzan M, Sun Y, Sullivan N, Rollins B, Ponath PD, et al. The beta-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates. Cell. 1996. 85:1135–1148.
3. Sullivan N, Sun Y, Sattentau Q, Thali M, Wu D, Denisova G, et al. CD4-Induced conformational changes in the human immunodeficiency virus type 1 gp120 glycoprotein: consequences for virus entry and neutralization. J Virol. 1998. 72:4694–4703.
4. Deng H, Liu R, Ellmeier W, Choe S, Unutmaz D, Burkhart M, et al. Identification of a major co-receptor for primary isolates of HIV-1. Nature. 1996. 381:661–666.
Article
5. Wu L, Gerard NP, Wyatt R, Choe H, Parolin C, Ruffing N, et al. CD4-induced interaction of primary HIV-1 gp120 glycoproteins with the chemokine receptor CCR-5. Nature. 1996. 384:179–183.
Article
6. Speck RF, Wehrly K, Platt EJ, Atchison RE, Charo IF, Kabat D, et al. Selective employment of chemokine receptors as human immunodeficiency virus type 1 coreceptors determined by individual amino acids within the envelope V3 loop. J Virol. 1997. 71:7136–7139.
Article
7. Feng Y, Broder CC, Kennedy PE, Berger EA. HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science. 1996. 272:872–877.
8. Zhu T, Mo H, Wang N, Nam DS, Cao Y, Koup RA, et al. Genotypic and phenotypic characterization of HIV-1 patients with primary infection. Science. 1993. 261:1179–1181.
9. Emini EA, Putney SD. Human immunodeficiency virus. Biotechnology. 1992. 20:309–326.
Article
10. Shioda T, Oka S, Ida S, Nokihara K, Toriyoshi H, Mori S, et al. A naturally occurring single basic amino acid substitution in the V3 region of the human immunodeficiency virus type 1 env protein alters the cellular host range and antigenic structure of the virus. J Virol. 1994. 68:7689–7696.
Article
11. Wolfs TF, Zwart G, Bakker M, Valk M, Kuiken CL, Goudsmit J. Naturally occurring mutations within HIV-1 V3 genomic RNA lead to antigenic variation dependent on a single amino acid substitution. Virology. 1991. 185:195–205.
Article
12. Fomsgaard A, Nielsen HV, Bryder K, Nielsen C, Machuca R, Bruun L, et al. Improved humoral and cellular immune responses against the gp120 V3 loop of HIV-1 following genetic immunization with a chimeric DNA vaccine encoding the V3 inserted into the hepatitis B surface antigen. Scand J Immunol. 1998. 47:289–295.
Article
13. Mrsny RJ, Daugherty AL, Fryling CM, FitzGerald DJ. Mucosal administration of a chimera composed of Pseudomonas exotoxin and the gp120 V3 loop sequence of HIV-1 induces both salivary and serum antibody responses. Vaccine. 1999. 17:1425–1433.
14. Lu Y, Xiao Y, Ding J, Dierich M, Chen YH. Immunogenicity of neutralizing epitopes on multiple-epitope vaccines against HIV-1. Int Arch Allergy Immunol. 2000. 121:80–84.
Article
15. Stover CK, de la Cruz VF, Bansal GP, Hanson MS, Fuerst TR, Jacobs WR Jr, et al. Use of recombinant BCG as a vaccine delivery vehicle. Adv Exp Med Biol. 1992. 327:175–182.
Article
16. Stover CK, de la Cruz VF, Fuerst TR, Burlein JE, Benson LA, Bennett LT, et al. New use of BCG for recombinant vaccines. Nature. 1991. 351:456–460.
Article
17. Choi BK, Cho SH, Bai GH, Kim SJ, Hyun BH, Choe YK, et al. Prevention of encephalomyocarditis virus-induced diabetes by live recombinant Mycobacterium bovis bacillus Calmette-Guérin in susceptible mice. Diabetes. 2000. 49:1459–1467.
Article
18. Connell ND, Medina-Acosta E, McMaster WR, Bloom BR, Russell DG. Effective immunization against cutaneous leishmaniasis with recombinant bacille Calmette-Guérin expressing the Leishmania surface proteinase gp63. Proc Natl Acad Sci U S A. 1993. 90:11473–11477.
Article
19. Li F, Horton H, Gilbert PB, McElrath JM, Corey L, Self SG. HIV-1 CTL-based vaccine immunogen selection: antigen diversity and cellular response features. Curr HIV Res. 2007. 5:97–107.
Article
20. Mölder T, Adojaan M, Kaldma K, Ustav M, Sikut R. Elicitation of broad CTL response against HIV-1 by the DNA vaccine encoding artificial multi-component fusion protein MultiHIV--study in domestic pigs. Vaccine. 2009. 28:293–298.
Article
21. Yang OO. Retracing our STEP towards a successful CTL-based HIV-1 vaccine. Vaccine. 2008. 26:3138–3141.
Article
22. Ratner L, Haseltine W, Patarca R, Livak KJ, Starcich B, Josephs SF, et al. Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature. 1985. 313:277–284.
Article
23. Honda M, Matsuo K, Nakasone T, Okamoto Y, Yoshizaki H, Kitamura K, et al. Protective immune responses induced by secretion of a chimeric soluble protein from a recombinant Mycobacterium bovis bacillus Calmette-Guérin vector candidate vaccine for human immunodeficiency virus type 1 in small animals. Proc Natl Acad Sci U S A. 1995. 92:10693–10697.
Article
24. Sell S, Hsu PL. Delayed hypersensitivity, immune deviation, antigen processing and T-cell subset selection in syphilis pathogenesis and vaccine design. Immunol Today. 1993. 14:576–582.
Article
25. Kohm AP, Sanders VM. Suppression of antigen-specific Th2 cell-dependent IgM and IgG1 production following norepinephrine depletion in vivo. J Immunol. 1999. 162:5299–5308.
26. Martin RM, Lew AM. Is IgG2a a good Th1 marker in mice? Immunol Today. 1998. 19:49.
27. Yu XF, Wang Z, Beyrer C, Celentano DD, Khamboonruang C, Allen E, et al. Phenotypic and genotypic characteristics of human immunodeficiency virus type 1 from patients with AIDS in northern Thailand. J Virol. 1995. 69:4649–4655.
Article
28. Zvi A, Kustanovich I, Hayek Y, Matsushita S, Anglister J. The principal neutralizing determinant of HIV-1 located in V3 of gp120 forms a 12-residue loop by internal hydrophobic interactions. FEBS Lett. 1995. 368:267–270.
Article
29. Isaka Y, Sato A, Miki S, Kawauchi S, Sakaida H, Hori T, et al. Small amino acid changes in the V3 loop of human immunodeficiency virus type 2 determines the coreceptor usage for CXCR4 and CCR5. Virology. 1999. 264:237–243.
Article
30. Brander C, Walker BD. T lymphocyte responses in HIV-1 infection: implications for vaccine development. Curr Opin Immunol. 1999. 11:451–459.
Article
31. Flynn JL. Recombinant BCG as an antigen delivery system. Cell Mol Biol (Noisy-le-grand). 1994. 40:Suppl 1. 31–36.
32. Shen Y, Shen L, Sehgal P, Huang D, Qiu L, Du G, et al. Clinical latency and reactivation of AIDS-related mycobacterial infections. J Virol. 2004. 78:14023–14032.
Article
33. Falk LA, Goldenthal KL, Esparza J, Aguado MT, Osmanov S, Ballou WR, et al. Recombinant bacillus Calmette-Guérin as a potential vector for preventive HIV type 1 vaccines. AIDS Res Hum Retroviruses. 2000. 16:91–98.
Article
34. McKinney DM, Skvoretz R, Livingston BD, Wilson CC, Anders M, Chesnut RW, et al. Recognition of variant HIV-1 epitopes from diverse viral subtypes by vaccine-induced CTL. J Immunol. 2004. 173:1941–1950.
Article
35. Berzofsky JA, Ahlers JD, Derby MA, Pendleton CD, Arichi T, Belyakov IM. Approaches to improve engineered vaccines for human immunodeficiency virus and other viruses that cause chronic infections. Immunol Rev. 1999. 170:151–172.
Article
36. Shibuya K, Robinson D, Zonin F, Hartley SB, Macatonia SE, Somoza C, et al. IL-1 alpha and TNF-alpha are required for IL-12-induced development of Th1 cells producing high levels of IFN-gamma in BALB/c but not C57BL/6 mice. J Immunol. 1998. 160:1708–1716.
37. Aguilar D, Infante E, Martin C, Gormley E, Gicquel B, Hernandez Pando R. Immunological responses and protective immunity against tuberculosis conferred by vaccination of Balb/C mice with the attenuated Mycobacterium tuberculosis (phoP) SO2 strain. Clin Exp Immunol. 2007. 147:330–338.
Article
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