MVA HIV-1 subtype C vaccine for India

Vaccine 24 (2006) 2585–2593

Development of a candidate DNA/MVA HIV-1 subtype C vaccine for India Sanjeev Kumar a , Priya Aggarwal a , Madhu Vajpayee a , R.M. Pandey b , Pradeep Seth a,c,∗ a b

Department of Microbiology, All India Institute of Medical Sciences, New Delhi 110029, India Department of Biostatistics, All India Institute of Medical Sciences, New Delhi 110029, India c Seth Research Foundation, D-1, Hauz Khas, New Delhi 110016, India

Received 12 June 2005; received in revised form 1 December 2005; accepted 7 December 2005 Available online 4 January 2006

Abstract Development of a vaccine against human immunodeficiency virus type-1 (HIV-1) is the mainstay for controlling the AIDS pandemic. An ideal HIV vaccine should induce neutralizing antibodies, CD4+ helper T cells, and CD8+ cytotoxic T cells. While the induction of broadly neutralizing antibodies remains a highly challenging goal, there are a number of technologies capable of inducing potent cellmediated responses in animal models, which are now starting to be tested in humans. Naked DNA immunization is one of them. The present study focuses on the stimulation cell-mediated and humoral immune responses by recombinant DNA–MVA vaccines, the areas where this technology might assist either alone or as a part of more complex vaccine formulations in the HIV vaccine development. Candidate recombinant DNA–MVA vaccine formulations expressing the human immunodeficiency virus-1 env and gagprotease genes from HIV-1 Indian subtype C were constructed and characterized. A high level of expression of the respective recombinant MVA (rMVA) constructs was demonstrated in BHK-21 cells followed by the robust humoral as well as cell mediated immune (CMI) responses in terms of magnitude and breadth. The response to a single inoculation of the rDNA vaccine was boosted efficiently by rMVA in BALB/c mice. This is the first reported candidate HIV-1 DNA/MVA vaccine employing the Indian subtype C sequences and constitutes a part of a vaccine scheduled to enter a preclinical non-human primate evaluation in India. © 2005 Elsevier Ltd. All rights reserved. Keywords: DNA vaccination; HIV-1 subtype C; DNA/MVA prime-boost

1. Introduction Viral diversity remains one of the greatest challenges for developing an effective HIV vaccine. The predominant subtype occurring in the India and southern African region is subtype C as well as in over 50% of all HIV-1 infections globally [1]. Strong correlates between strong cell mediated immune (CMI) response and long-term non-progressors have suggested the possible use of vaccine inducing CMI [2,3]. An obvious approach for establishing strong cellular immu∗ Corresponding author. Tel.: +91 11 51044445; fax: +91 11 51656975; mobile: +91 9810311407 E-mail addresses: [email protected], [email protected] (P. Seth).

0264-410X/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2005.12.032

nity to specific pathogens is through repeated vaccination. The idea of ‘boosting’ immune responses has been around as long as vaccines and repeated administrations with the same vaccine (homologous boosting) have proven very effective for boosting humoral responses. However, this approach is relatively inefficient at boosting cellular immunity because prior immunity to the vector tends to impair robust antigen presentation and the generation of appropriate inflammatory signals. One approach to circumvent this problem has been the sequential administration of vaccines (typically given weeks apart) that use different antigen-delivery systems (heterologous boosting). The basic prime-boost strategy involves priming the immune system to a target antigen delivered by one vector and then selectively boosting this immunity by re-administration of the antigen in the context of a distinct

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second vector. The key strength of this strategy is that greater level of immunity is established by heterologous prime-boost regimen, than cannot be attained by a single vaccine administration or homologous boost strategies. With some of the early prime-boost strategies this effect was merely additive, whereas with some of the newer strategies (usually involving poxvirus or adenovirus boosting) powerful synergistic effects are achieved [4,5]. This synergistic enhancement of immunity to the target antigen is reflected in an increased number of antigen-specific T cells, selective enrichment of high avidity T cells and increased efficacy against pathogen challenge. Vaccination strategies in which a DNA prime is boosted with a poxvirus vector are particularly effective and have emerged as the predominant approach for eliciting protective CD8+ T-cell immunity [6]. This approach couples the strong priming (but poor boosting) properties of DNA vaccines with the strong boosting properties of vaccines based on viral vectors. The HIV-1 scenario in India is alarming with nearly 5.2 million people infected with the virus [7]. More than 90% of these infections are due to clade C viruses [8], which have cell tropism as well as pathogenicity different from subtype B viruses [9–12]. Therefore, it is prudent to make a candidate vaccine using locally circulating strain in India. In sight of the above factors, this study targeted envelope and gag proteins of HIV-1 Indian subtype C for the candidate vaccine formulations. We had earlier reported the construction and efficient expression of the recombinant MVA (rMVA) constructs (MVA gagprotease49587 and MVASK3) [13,14]. In this study, homologous (rDNA/rDNA, rMVA/rMVA) and heterologous (rDNA/rMVA) prime-boost regimens were compared in BALB/c mouse model. The recombinant MVA constructs were found to be highly immunogenic tool in comparison to rDNA constructs alone, as judged by the elicitation of the antibody production and induction of robust T-cell responses. In addition, these recombinant live viral vector based vaccine constructs were not only found to be immunogenic but also important tool for the DNA/MVA prime-boost based immunization regimen.

2. Materials and methods 2.1. Cells BHK-21 (Clone 13) cell line, obtained from National Center for Cell Science (Pune, India) was grown and maintained MEM (E) medium with 10% fetal calf serum (Sigma, USA).

protease (Genbank # AF533140) genes, respectively in plasmid vector pJW4304 (kind gift of Dr. Jim Mullins, Univ. of Washington, Seattle) as described earlier [15–17]. The resultant DNA vaccine constructs pJWSK3 (6.5 kb) of envgp120 gene and pJWgagprotease49587 (7.1 kb) were used in this study. Modified Vaccinia Ankara (MVA) was a kind gift from Dr. Bernard Moss, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland. Construction and immunogenicity testing of two recombinant MVA containing same HIV-1 subtype C envelope gp120 and gagprotease genes, respectively have been reported earlier from our laboratory [13,14]. rMVA viruses were grown in BHK-21 cells and titrated by plaque assay. For immunization, both rMVAs were purified on 36% sucrose cushion (10 mM Tris–Cl, pH9.0) as described earlier [14]. 2.3. Peptides 2.3.1. Gag peptides A set of 49 peptides each containing 20 amino acid (aa) residues (20 mer) overlapping by 10 aa representing the entire aa sequence of gag protein of HIV-1 from subtype C (Catalogue # 3933), were obtained from NIH AIDS Research Reference Reagent Program (NIH, Bethesda, MD). Each peptide was dissolved in dimethyl sulphoxide (DMSO) (Sigma, USA) at 20 mg/ml. These peptides were pooled in a matrix format into 14 pools with each pool containing 7 peptides at a final concentration of 2 ␮g/ml per peptide so that there were 7 column pools (C1–7) and 7 row pools (R1–7) (Fig. 1). Each peptide was represented twice, once in column pool and once in row pool. Each pool was tested for interferon ␥ production from splenocytes. The peptides at the intersection of the positive row and positive column pools were identified as the peptides responsible for stimulation of splenocytes from immunized mice. These peptides were then checked individually in duplicate for confirmation. 2.3.2. Envelope peptides A set of 125 peptides each containing 15±1 aa residues overlapping by 11 aa, which represented the entire protein of envelope gp120 of HIV-1 from subtype C consensus was synthesized Bio-Synthesis Incorporated (TX, USA). These peptides were pooled into 5 pools (P1–5) with each pool containing 25 continuous peptides at a final concentration of 2 ␮g/ml/peptide. Each pool was tested for interferon ␥ production from splenocytes by ELISpot assay (Fig. 2). These peptides were not tested in matrix format.

2.2. Vaccine constructs 2.4. Immunization studies in BALB/c mice Peripheral blood mononuclear cells from two HIV-1 seropositive, asymptomatic, ART (anti-retroviral therapy) na¨ıve individuals (Lab ID # 49486 and 49587) with CD4+ Tcell count of >500 cells/mm3 , plasma virus load of <500/ml infected with HIV-1 subtype C were processed for molecular cloning of envelope gp120 (Genbank #AY775283) and gag-

Four to six weeks old female BALB/c mice were purchased from National Central Laboratory Animal Sciences (NCLAS), National Institute of Nutrition, Hyderabad, India. Experimental protocol was approved by: (i) Institutional Biosafety Committee for Recombinant DNA Molecules, and

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Fig. 1. Pooling of 49 overlapping gag peptides from HIV-1 subtype C in a matrix format for the evaluation of cell mediated immune response by IFN-␥ ELISpot assay. Region-wise distribution of overlapping peptides on the Gag protein is diagrammatically shown below the table.

(ii) Institutional Small Animal Ethics Committee as per the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals, Government of India. There were five mice in each experimental group. Mice were primed intradermally with 107 pfu of either recombinant MVA construct (MVAgagprotease49587 or MVASK3) or 50 ␮g of either recombinant plasmid DNA (rDNA) construct (pJWgagprotease49587 or pJWSK3) in 50 ␮l normal saline using 27-gauge needle as per immunization regimen (Fig. 3). Homologous boosting (DNA/DNA, rMVA/rMVA) or the heterologous boosting (DNA/rMVA) of the primed mice with the same dose of the vaccinogens were followed

2 weeks after the priming dose. Control mice received MVA vector alone (vector control) or normal saline intradermally. Mice were bled thrice from caudal vein at 0, 2 and 4 weeks time points as shown in Fig. 3. Mice were sacrificed with overdose of pentobarbital 2 weeks after the priming as well as boosting dose according to immunization schedule. The spleens were removed aseptically and the cells were collected and resuspended in RBC lysis buffer to remove erythrocytes. The cells from the same group of mice were pooled and resuspended in RPMI with 10% FCS and enumerated. The humoral immune response was evaluated by ELISA using serum and the cell mediated immune response by IFN-␥

Fig. 2. Pooling of 125 continuous overlapping envelope gp120 peptides from HIV-1 subtype C into 5 pools (P1–5) of 25 peptides/pool for the evaluation of cell mediated immune response by IFN-␥ ELISpot assay. Diagram below the table shows region-wise distribution of peptides on Env gp120.

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Fig. 3. Schematic of mice immunization schedule. Immunization studies for envgp120 and gagprotease constructs were carried out separately utilizing same vaccination regimen as shown in group I–V. Mice were immunized intradermally twice, either with DNA/DNA, MVA/MVA or DNA/MVA with the dose of 50 ␮g/mouse or 107 pfu/mouse at week 0 and 2. There were five mice per group. Mice in group-VI were inoculated with normal saline. Mice were bled at weeks 0, 2 and 4 for the detection of HIV-1 specific binding antibodies. At week 2 and 4 (2 weeks after priming and boosting dose) mice were sacrificed to obtain spleens for doing IFN-␥ ELISpot assay.

ELISpot assay with pooled splenocytes from each group of mice. 2.5. ELISA Serum samples obtained from mice just prior to immunization, at the end of second week following first dose and then 2 weeks after the booster were assayed for antibody by ELISA using plates coated with 10 ng of HIV-1 virus lysate. Preimmune sera were used as the control. The plate was read at O.D. 450 nm on Labsystems Multiskan Plus (Finland) ELISA plate reader. The cut-off (CO) value was calculated by the following formula: Cut-off (CO) = Mean O.D. of preimmune sera from all groups of mice + 3S.D. Any serum sample giving higher O.D. than the CO was taken as positive for HIV antibodies. 2.6. IFN-γ ELISpot assay Spleen cells from immunized mice were assayed for their ability to secrete IFN-␥ during in vitro restimulation assay with antigenic peptides using a modified ELISpot assay [18]. Murine IFN-␥ ELISpot capture and development modules were purchased from R&D systems (USA) and immunoassay was put up as per the manufacturer’s instructions. Assay was performed as follows. Ninety-six well PVDF backed plates (Millipore, Bedford, MA) were coated with antimurine IFN-␥ capture antibody overnight at 4 ◦ C and subsequently blocked in 1% BSA and 5% Sucrose in PBS (pH7.4)

for two hours at room temperature. The plates were then incubated with complete RPMI medium containing 10% FCS for half an hour. Splenocytes, at a seeding concentration of 5 × 105 /well, were incubated with each peptide pool (2 ug each peptide/ml) from either gag region or envelope gp120 region. After overnight incubation at 37 ◦ C and 5% CO2, the plates were washed five times with wash buffer (PBS + 0.05%Tween 20). Plates were then incubated with biotinylated anti-mouse IFN-␥ detection antibody (R&D Systems) at 4 ◦ C overnight. Thereafter, the plates were washed and incubated with Streptavidin-AP conjugate for two hours at room temperature. After four washes, spots were developed with BCIP/NBT reagent (R&D Systems). Finally, spots were counted using ELISpot plate reader (Zeiss KS ELISpot, Germany) by capturing the images of individual wells. Negative control wells included DMSO in RPMI medium, PHA at a concentration of 2 ␮g/ml was used as a positive control. Cut-off value was calculated by the following formula: Cut-off = Mean of spots in three negative control wells + 3S.D. Wells showing greater number of spots than the cut-off value were taken as positive. 2.7. Statistical analysis Quantitative variables were summarized by mean and standard deviation. Student’s t-test was used to compare frequencies amongst the groups. In this study, p < 0.05 has been considered statistically significant.

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Fig. 5. Cell-mediated responses by immunizing HIV-1 gagprotease constructs using IFN-␥ ELISpot assay. The response elicited at 4 weeks time point (2 weeks after the booster dose) is expressed as spot forming units (SFU) per million splenocytes. The assay was done with 20 gag peptides (shown in the graph), which were worked out from the 14 matrix peptide pools and shown to be immunogenic in the initial screening assay. Dotted line represents the cut-off value as calculated by mean of spots in three negative control wells +3S.D.

Fig. 4. (a) Antibody response by immunizing HIV-1 gagprotease constructs. Groups of five female BALB/c mice each, were immunized with recombinant gag protease DNA and MVA constructs to perform DNA/DNA, MVA/MVA and DNA/MVA prime-boost regimen according to the immunization schedule described in the text. Shown are anti-gag antibodies, as measured by ELISA, plotted as mean absorbance O.D. values @ 450 nm. Error bars indicate standard error of the mean O.D. values. (b) Antibody responses by immunizing HIV-1 envelope gp120 constructs. Groups of five female BALB/c mice each, were immunized with recombinant envgp120 DNA and MVA constructs to perform DNA/DNA, MVA/MVA and DNA/MVA primeboost regimen according to the immunization schedule shown in Fig. 1. Shown are anti-env gp120 antibodies, as measured by ELISA, plotted as mean absorbance O.D. values @ 450 nm. Error bars indicate standard error of the mean O.D. values.

rDNA/rMVA or rMVA/rMVA protocol showed significantly higher absorbance (1.034 ± 0.142 and 2.3 ± 0.307, respectively) at 4 weeks time point as compared to the absorbance of 0.124 ± 0.025 in mice immunized with env gp120 vaccine constructs by rDNA/r DNA protocol (p < 0.005, Fig. 4b). Priming and boosting with rMVA constructs (rMVA/rMVA) produced significantly higher antibody levels as compared to rDNA/rMVA immunization protocol (p < 0.05). 3.2. Cell mediated immune response by IFN-γ ELISpot assay The data presented in Tables 1 and 2 and Figs. 5 and 6 represent combined response of CD4+ and CD8+ T-cell responses. Mice immunized with gagprotease gene constructs by rDNA/rDNA protocol induced an average of 74

3. Results 3.1. Humoral immune response by ELISA The absorbance of preimmune serum samples from all the groups did not vary significantly and therefore, all the absorbance values were pooled to calculate the cut-off value (mean = 0.192 ± 0.116; cut off = 0.540). The data presented in Fig. 4a show that the sera from mice immunized with gagprotease constructs by either rDNA/rMVA or rMVA/rMVA protocols recorded significantly higher absorbance (1.06 ± 0.01 or 1.6 ± 0.15, respectively) than the cut-off value suggesting induction of gag specific antibody response as compared to that from mice immunized with rDNA (pJWgagprotease49587)/rDNA (pJWgagprotease49587) protocol (0.369 ± 0.124) at 4 weeks time point (p < 0.005). Similarly, sera from mice immunized with envelope gp120 gene constructs by either

Fig. 6. Cell-mediated responses by immunizing HIV-1 gp120 constructs using IFN-␥ ELISpot assay. The response elicited at 4 weeks time (2 weeks after the booster dose) is expressed as spot forming units (SFU) per million splenocytes. The assay was done using five envelope HIV-1 subtype C peptide pools (P1–5) comprising 125 peptides. Dotted line represents the cut-off value as calculated by mean of spots in three negative control wells +3S.D.

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Table 1 Evaluation of CTL responses induced by immunization of mice with HIV-1 gag protease candidate vaccinogens by IFN-␥ ELISpot assay after priming and boosting Immunization groups

Priming immunogen

Reactive gag peptides

Reactive gag regions

Range of SFU/106 PBMCs (Mean ± S.D.)

Boosting immunogen

Reactive gag peptides

Reactive gag regions

rDNA/rDNA

rDNA

No Response

–

–

rDNA

P17, P24, P2, P7, P1, P6

42–130 (74 ± 28.21)

rMVA/rMVA

rMVA

2, 3, 15, 17, 19, 23, 29, 43, 45, 47

P17, P24, P7, P1, P6

100–170 (126 ± 18)

rMVA

P17, P24, P2, P7, P1, P6

100–204 (143 ± 29.87)

rDNA/rMVA

rDNA

No Response

–

–

rMVA

8–10, 15, 16, 29, 31, 33, 37, 43, 45, 47 1–3, 5, 8–10, 12, 15–17, 19, 23, 29, 31, 33, 37, 43, 45, 47 1–3, 5, 8–10, 12, 15–17, 19, 23, 29, 31, 33, 37, 43, 45, 47

P17, P24, P2, P7, P1, P6

130–446 (301 ± 99.4)

Range of SFU/106 PBMCs (Mean ± S.D.)

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Table 2 Evaluation of CTL responses induced by immunization of mice with HIV-1 env gp120 candidate vaccinogens by IFN-␥ ELISpot assay after priming and boosting Immunization groups

Priming immunogen

Reactive envelope pools

Reactive envelope regions (estimated)

Range of SFU/106 PBMCs (Mean ± S.D.)

Boosting immunogen

Reactive envelope pools

Reactive envelope regions (estimated)

Range of SFU/106 PBMCs (Mean ± S.D.)

rDNA/rDNA rMVA/rMVA

rDNA rMVA

No response P1, P2, P3, P4

– 92–156 (127 ± 26)

rDNA rMVA

P1, P3, P4 P1, P2, P3, P4

C1, C2, C3, V3 C1, C2, C3, V1, V2, V3

36–82 (52 ± 25) 154–222 (180 ± 30)

rDNA/rMVA

rDNA

No response

– C1, C2, C3, V1, V2, V3 –

–

rMVA

P1, P2, P3, P4

C1, C2, C3, V1, V2, V3

134–142 (140 ± 3.6)

S. Kumar et al. / Vaccine 24 (2006) 2585–2593

SFU ± (28)/106 splenocytes (range of 42–130 SFU/106 cells) at 2 weeks after the boosting dose. A total of 12 peptides from different regions of gag were found reactive in these mice (Table 1). Whereas, spleen cells from mice immunized with gagprotease gene constructs by rDNA/rMVA or rMVA/rMVA protocols produced 301 SFU/106 cells (range 130–446 SFU/106 cells) and 143 SFU/106 cells (range 100–204 SFU/106 cells), respectively. Twenty peptides were found to be reactive from different regions of gag with either of these immunization schedules (Fig. 5). Similarly, in assay with spleen cells from mice immunized with env gp120 gene constructs by rDNA/rDNA protocol, three out of five pools (P1, P3 and P4) of envelope peptides encompassing C1, C2, C3, and V3 regions of gp120 were found to be reactive, producing SFUs ranging from 36 to 82/106 cells (average 52 SFU/106 ; Table 2). Similarly, in mice immunized with either rDNA/rMVA or rMVA/rMVA protocols, four out of five pools (P1, P2, P3 and P4) were found to be reactive in ELISpot assay with splenocytes producing an average of 140 SFU/106 cells (range 134–142 SFU/106 cells) and 180 SFU/106 cells (range 154–222 SFU/106 cells), respectively (Table 2 and Fig. 6). Splenocytes from mice in group VI were not secreting IFN-␥ and served as control group. 4. Discussion In this study, we evaluated immunogenicity of rDNA and rMVA vaccine constructs encoding HIV envelope Gp120 and Gagprotease in murine model as homologous and heterologous prime-boost protocols. Prime-boost immunization strategy is effective at generating high levels of T-cell memory. The basic strategy involves priming the immune system to a target antigen delivered by one vector and then selectively boosting the immunity generated by re-administration of the antigen in the context of a second and distinct vector. This approach couples the strong priming (but poor boosting) properties of DNA vaccines with the strong boosting properties of vaccines based on viral vectors. Vaccination strategies in which a DNA prime is boosted with a poxvirus vector are especially effective and have emerged as the predominant approach for eliciting protective CD8+ T-cell immunity. Although much of the early work using this strategy was driven by efforts to develop vaccines to control malaria, it was subsequently applied to vaccine development against a variety of pathogens. Recently, several studies have demonstrated the efficacy of prime-boost vaccination strategies in generating cellular immunity to a variety of pathogens. These include SIV/HIV [19,20], malaria [21], hepatitis C virus, M. tuberculosis and hepatitis B virus [22]. Although in most immunogenicity studies in animal models with rDNA or rMVA vaccine constructs intramuscular has been a preferred route of immunization because of ease of injecting the immunogen over the intradermal route in which the immunogen is required to be injected at multiple sites, we

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employed intradermal route of immunization. Striated muscle cells and skin cells have been shown to take up and express free DNA in the absence of viral vector or physical carrier [23]. Skin and mucous membranes are rich in antigen presenting cells. Muscle is not considered to be a good site for antigen presentation, as it contains few if any dendritic cells, macrophages and lymphocytes [24,25]. Interestingly, though Shiver et al. found that intramuscular injection of mice gives a better CMI response than with intradermal immunization [26,27], other studies have observed a comparable or even higher levels of immune response following intradermal route than intramuscular route of immunization [28,29]. We compared the efficacy of heterologous prime-boost regimen (in which priming was done with rDNA constructs and boosting was done with rMVA constructs, rDNA/rMVA) with that of homologous prime-boost regimen (in which both priming and boosting were done with either rDNA constructs, rDNA/rDNA, or with rMVA constructs, rMVA/rMVA) in induction of immune response in mice. Interestingly, priming dose with either plasmid DNA construct, pJWgagprotease49587 or pJWgp120 did not induce sufficient CTL immune response after 2 weeks of priming dose to be detected by ELISpot assay (Tables 1 and 2) whereas priming with either rMVA (MVAgagprotease 49587 or MVAgp120) induced moderately good CTL response at the same time point (126 or 127 SFU/106 cells). Asakura et al. [30] have also observed a failure of generation of cytotoxic T-cells after the single dose of DNA immunization. However, following boosting with rMVA we observed that there was a tremendous increase in the number of epitope recognition and in the number of IFN-␥ secreting cells by ELISpot assay particularly after heterologous prime-boost regimen (rDNA/rMVA) as compared to either homologous prime-boost regimens (Fig. 5). DNA alone has been shown to stimulate weak CD8+T cell response in macaques, but primes the immune cells for subsequent responses to a recombinant viral vaccine that are better than the response to either vaccine alone [31–34]. Similarly, rDNA/rMVA prime-boost regimens of either gagprotease or envgp120 vaccine constructs induced 7–8fold higher antibody response in mice as compared to rDNA/rDNA regimen (Fig. 4a and b). Priming dose of either rDNA construct induced very low amount of antibodies. However, the humoral response evaluation was restricted to binding antibodies only since neutralizing antibody assays do not give consistent results with mouse sera (David Montefiori, personal communication). In a previous study from our laboratory, immunization with repeated doses of DNA envelope construct failed to elicit humoral response significantly [15]. Indicating thereby, the DNA construct was immunogenic but could not elicit the antibody formation to detectable levels. Alternatively, the poor immune response may be due to insufficient amount of envelope expression by the DNA constructs [35,36] boosting with rMVA after rDNA priming is likely to result in higher levels of antigen expression, which induce higher levels of binding antibodies. Similar observations were

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also made by Amara et al. [37], where they could get a 10fold higher level of binding antibodies for the envelope region with rDNA/rMVA immunization strategy [37]. To summarize in the present study both MVA constructs (MVA gagprotease49587 and MVASK3) were found to be highly immunogenic tool for the prime-boost regimen, as they could elicit the antibody production and induced Tcell population. The recombinant live viral vector based vaccine constructs not only were found to be immunogenic but also important tool for the DNA/MVA prime-boost based immunization regimen. Nonetheless, this study on potential HIV-1 Indian subtype C based vaccine strategies is limited to demonstration of immunogenicity in murine model only.

[11]

[12]

[13]

[14]

[15]

Acknowledgement We gratefully acknowledge support by the research grant from the Department of Biotechnology, Ministry of Science and Technology, Govt. of India, under Prime minister’s, Jai Vigyan Mission Program. Sanjeev Kumar was the recipient of fellowship from University Grants Commission (UGC), India. Priya Aggarwal was recipient of fellowship from Council of Scientific and Industrial Research (CSIR), India SRF fellowship program.

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