The Immune Responses Of The Primeboost Regimen With rBCGE12 AND rDISE12 Candidate Vaccine

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P. Leangaramgul(1), S. Sapsutthipas(1), K. Balachandra(1), K. Matsuo(2), T. Hamano(2), M. Honda(2)

(1) Medical Biotechnology Center, Department of Medical Sciences, Ministry of Public

Health, Tivanon Rd., Muang, Nonthaburi, 11000, Thailand. (2)AIDS Research Center,

National Institute of Infectious Diseases, Shinjuku-ku, Tokyu 162-8640, Japan

Abstract: In the previous study, a research group in NIID, Japan demonstrated that the V3 sequence of 12 amino acids of HIV-1 CRF01_AE (E12 epitope) fused with mycobacterial a-antigen was secreted from BCG cells (rBCG-E12) and could induce NT-Ab against CRF01_AE primary isolates. However, the NT-Ab titer in guinea pig was not enough to obtain protective efficacy. So, we attempted to boost the NT-Ab by rDIs expressing E12 epitope-a-antigen fusion protein (rDIs-E12).

We have started the second generation AIDS vaccine research project and successfully constructed rDIs-E12 which expressed E12 epitope under control of vaccinia p7.5 promoter in infected chicken embryo fibroblast cell. The rBCG-E12 clone that could secrete E12 epitope under control of a-antigen promoter was provided us by NIID, Japan group. The expression of a-antigen-E12 fusion protein in the both rDIs-E12 and rBCG-E12 were checked by Western blot analysis using HIV+ human serum and the size of the fusion protein was approximately 32 kDa. To test immunogenicity, we primed Balb/c mice with rBCG-E12 and boosted with rDIs-E12 to analyze effect of prime-boost regimen for NT-Ab production. The ELISPOT was performed for the quantitation of antigen-specific CD8 T cells responses. We found that the E12 itself could not stimulate CTL response, it might not be CTL epitope in Balb/c mice. The rBCG and rDIs vectors could stimulate CTL activity, this means CTL activity was induced with alpha antigen and PPD. The CTL responses increase after second boosting with rDIs. The ELISA was also performed for check the antibody titer against E12 and alpha antigen. There are antibody titer against alpha antigen but no antibody titer against E12 peptide. From the experiment, we conclude that priming with rBCG-E12 and boosting with rDIs-E12 could stimulate CTL responses and have boosting effect but could not stimulate antibody titer. Further study will develop the regimen of immunization and using new epitope for candidate vaccine.

Key words: HIV, Vaccine, Immune response, CTL, BCG, Vaccinia.


The development of an effective prophylactic vaccine for human immunodeficiency virus type 1 (HIV-1) is hindered by the lack of a known immunologic correlate of protection. However, until an efficacy trial is undertaken, those evaluating candidate vaccines must rely on criteria chosen from other clinical settings, such as the immune responses found in HIV-1 infected long-term nonprogressors and in HIV-1 exposed but uninfected, and primate vaccine studies. It is widely believed that two of more important responses for preventing or controlling HIV infection are a vigorous CD8+ cytotoxic T lymphocyte (CTL) response (CD8+ CTL) and the development of antibody that would neutralize primary transmitted viruses (1-4).

Genetic diversity in HIV-1 is well evidenced from the large number of different HIV-1 strain isolated around the world, which have been divided into three groups. The major group has been further divided into 10 nucleotide sequence defined subtypes (5, 6). In Thailand, two major subtypes of HIV-1 are prevalent: clade B' (Thai subtype B) in intravenous drug abusers and clade E (Thai subtype A) in heterosexuals (7, 8).

Most of the candidate vaccines currently in production are based on B clade virus which, although prevalent in the developed countries, is not the clade which is found in most parts of the developing countries. HIV-1 clade B-derived vaccine could hardly prevent infection of clade E virus (9).

Recombinant live attenuated Mycobacterium bovis BCG (BCG) vector-based vaccine targeted to HIV-1 and simian immunodeficiency virus were reported to induce both humoral and cellular immune responses in animal models against a variety of antigens such as Gag, Env and Nef (10-14). The BCG immunization is known to generate primary Th1 and delayed-type hypersensitivity responses that are considered to be suitable for a vaccine development for HIV-1.

Recombinant viral vectors are believed to have similarlity to live attenuated vaccines. The expression vector results in antigen processing through the major histocompatibility (MHC) class I pathway, which induces CD8+ CTL. Earlier studies have evaluated the safety and immunogenicity of recombinant vaccinia vector (15, 16).

The objectives of this study are to construct the new candidate HIV-1 subtypeE vaccine, rBCG-E12 and rDIs-E12 and to develop a prime-boost strategy with the goal of eliciting broadly neutralizing antibodies against HIV-1 to provide sterilizing immunity for this virus.


I Design and construction of recombinant vaccinia virus (rDIs-E12) and recombinant BCG (rBCG-E12)

E12 epitope sequence was synthesized from the 12 amino acids (aa) E12 consensus sequence of HIV subtype E and inserted into the XhoI site of alpha-antigen in pUCvvp7.5-tPA in which a signal sequence of tissue plasminogen activator gene drives secretion of alpha antigen-E12 fusion protein. A fragment of p7.5-tPA-alpha K-E12 was inserted into the HindIII site of pUC/DIs as transfer vector.

Recombinant vaccinia virus (rDIs) was constructed by using this transfer vector and rDIsLacZ as the parental virus. The recombinant viruses were propagated in Chicken Embryo Fibroblast (CEF) cells.

Infection was performed onto CEF cells grown in 6-wells plate. Transfection was performed with 20 |lg of pUC/DIs/p7.5-tPA-alpha K-E12 using CLONFECTIN (Clontech). Recombinant DIs virus expressing E12 epitope was selected by two consecutive rounds as colorless plaques when stained with X-gal. The recombinant clones were grown in CEF cell cultures and checked the expression by Western blot analysis.

The recombinant BCG-E12 (rBCG-E12) that could secrete E12 epitope under control of a-antigen promoter was provided by NIID, Japan group. A fragment of aK antigen-E12 was inserted into the shuttle vector, pSO246 which was stable in BCG and then transformed into BCG Tokyo strain by electroporation with kanamycin resistance selection. The rBCG-E12 clone was checked the expression by Western blot analysis (18).

II Evaluation of the vaccine candidate by animal model

An optimal immune induction would be obtained by a prime-boost regimen of vaccination. The mice groups are shown in Table1. The balb/c mice were administered intradermal with rBCG-E12 or rBCG-a antigen. Eight weeks after inoculation, three mice of each group were sacrificed to assess humoral and cellular immune responses. The other mice were boosted with rDIs-E12 at 2 months after first inoculation. Two weeks after boosting three mice of each group were sacrificed for immune response checking and the remained mice were boosted with rDIs-E12 for further stimulate immune responses. Two weeks after final inoculation, all mice were sacrificed to assess immune responses.

The assessments of CTL responses were detected with gamma interferon by using the ELISPOT technique. The antibody responses were checked by ELISA for anti-V3 peptide (E12 peptide) and anti-alpha antigen.


I Design and construction of rDIs-E12 and rBCG-E12 candidate vaccine

A synthetic DNA fragment encoding the 12 amino acids (aa) V3 sequence of clade E consensus HIV (E12 epitope) was inserted into the Xhol site of alpha-antigen in pUCvvp7.5-tPA in which a signal sequence of tissue plasminogen activator gene drives secretion of alpha antigen-E12 fusion protein. A fragment of p7.5-tPA-alpha K-E12 about 2.3 kb was cloned into the HindIII site of pUC/DIs (4.7 kb) as transfer vector. The positive clone was selected by HindIII cut and obtained only one clone (#3) as shown in Figure 1.

These transfer vectors, pUC/DIs-E12 that harbored E-12 epitope gene in the cloning site of pUC/DIs, were used for the construction of recombinant DIs using rDIs-LacZ as the parental virus. We obtained recombinant vaccinia virus (rDIs-E12) that selected from colorless virus plaques stained with X-gal, purified two times by repetitive selection, and propagated in CEF cells.

Figure 1. Agarose gel electrophoresis analysis of pUC/DIs-E12 16 clones, which were digested with HindIII. Clone #3 contained 2.3 kb a-K antigen E12 gene and 4.7 kb pUC/DIs plasmid. M was represented for 1 kb DNA ladder.

II Expression and characterization of rDIs-E12 and rBCG-E12

The expression of alpha antigen E12 epitope fusion protein from rDIs-E12 and rBCG-E12 were checked by Western blot analysis using HIV+ human serum as primary antibody. We found that the alpha-K E12 fusion protein could express in culture supernatant of rDIs-E12 and shown molecular weight of approximately 32

Figure 2. Western blot analysis of a-K antigen-E12 fusion protein from rDIs-E12 using HIV+ human serum as primary antibody. Lanes 1-5; Cell lysate, Lanes6-10; Culture supernatant. Lanes1,6; rDIs-E12 #1-1, Lanes2,7; rDIs-E12 #1-2, Lanes3,8; rDIs-E12 #2-1, Lanes4,9; rDIs-E12 #4-1, Lanes5,10; rDIs-E12 #4-2. The positive bands of a-K antigen E12 were shown molecular weight approximately 32 kDa in culture supernatant.

kDa as shown in Figure 2.

kDa as shown in Figure 2.

III Evaluation of the vaccine candidate by animal model ELISPOT

Since IFN-y is a crucial component of antimycobacterial immunity, the induction of this cytokine in both the candidate vaccine and vector control of immunized mice was quantitatively assessed by the ELISPOT technique. The rBCG prime and rDIs-E12-boost group showed significant enhancement of ELISPOT activity against PPD (containing alpha antigen) as shown in Figure3. However, they did not show any significant activity against E12 peptide (Figure 4), indicating that V3 epitope of CRF01_AE did not act as a CTL epitope in Balb/c mice.

Figure 3. CTL activity against PPD: 1 = priming, 2 = first boosting, 3 = second boosting

Figure 4. CTL activity against E12 compared with ConA (positive control); mice groups 1-6 are shown in Table 1


Using an enzyme-linked immunosorbent assay (ELISA), the antigen specific E12 peptide, alpha-T antigen, and alpha-K antigen antibody titers were determined for sera from vaccinated animals as shown in Table1. There were antibody titer against alpha-T antigen and alpha-K antigen in all groups of mice but no antibody titer against E12 peptide.

Table 1. Humoral response to E12 peptide, alpha-T antigen, and alpha-K antigen in vaccinated mice.

Mice Group

Antibody Titer









Candidate Vaccine

1 2 3

rBCG-E12 rBCG-E12 + rDIs-E12 rBCG-E12 + rDIs-E 12 + rDIs-E12

500 500 UC

2000 2000 4000

1000 2000 2000

rBCG control


rBCG-pSO246 rBCG-pSO246 + rDIs-LacZ


4000 4000

2000 1000


rBCG-pSO246 + rDIs-LacZ + rDIs-LacZ









rDIs control


rDIs-E 12





rDIs-E12 + rDIs-E12



UC - Under Cut-off


In the present study, we have successfully constructed rDIs-E12 and rBCG-E12 candidate vaccines. To clarify the enhancement of HIV-1 specific immune response by the consecutive vaccine regimen using these two vaccine constructs, we have examined E12-specific ELISPOT (cellular immunity) and antibody production in mice.

From the ELISPOT experiments, the prime-boost regimen enhanced effector cell response for alpha antigen but could not enhance that for E12 peptide. Although the V3 epitope in HIV subtype B contains CTL epitope restricted by mouse class I H2d (17), to our knowledge, there is no report on such CTL epitope in HIV CRF01_AE,

CTL response against E12

CTL response against E12



1 n 1 1

and the lack of CTL induction should be ascribed to not matching class I-restriction in the HIV-1 subtype. However, the ELISPOT response against alpha antigen was significantly boosted by rDIs-E12, suggesting that the prime-boost regimen could have an effect for enhancing cellular immunity.

Regarding antibody response, rBCG-E12 immunization induced NT antibody production in guinea pigs (18) and in mice (data not shown). However, the rDIs-E12 boosting could not enhance anti-E12 peptide antibody titer. Taking account of enhancing anti-alpha antigen antibody titer by the same regimen, the antigenicity of E12 peptide inserted in rDIs-E12 was not enough for enhancing anti-E12 antibody, implying that the conformation of E12-alpha antigen fusion protein in rDIs-E12 may be different from that in rBCG-E12. To produce NT antibody for better HIV vaccine development, it should be tested to construct and evaluate such prime-boost regimen for the other Env construct of both rBCG and rDIs vector systems.


This work was supported by Japan Foundation for AIDS Prevention (JFAP) and Small Grants Program from International Society for Infectious Diseases (ISID), USA.


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