Depleting T regulatory cells by targeting intracellular Foxp3 with a TCR mimic antibody.

Depletion of T regulatory cells (Tregs) in the tumor microenvironment is a promising cancer immunotherapy strategy. Current approaches for depleting Tregs are limited by lack of specificity and concurrent depletion of anti-tumor effector T cells. The transcription factor forkhead box p3 (Foxp3) plays a central role in the development and function of Tregs and is an ideal target in Tregs, but Foxp3 is an intracellular, undruggable protein to date. We have generated a T cell receptor mimic antibody, "Foxp3-#32," recognizing a Foxp3-derived epitope in the context of HLA-A*02:01. The mAb Foxp3-#32 selectively recognizes CD4 + CD25 + CD127low and Foxp3 + Tregs also expressing HLA-A*02:01 and depletes these cells via antibody-mediated cellular cytotoxicity. Foxp3-#32 mAb depleted Tregs in xenografts of PBMCs from a healthy donor and ascites fluid from a cancer patient. A TCRm mAb targeting intracellular Foxp3 epitope represents an approach to deplete Tregs.

A novel antibody-TCR (AbTCR) platform combines Fab-based antigen recognition with gamma/delta-TCR signaling to facilitate T-cell cytotoxicity with low cytokine release.

The clinical use of genetically modified T-cell therapies has led to unprecedented response rates in leukemia and lymphoma patients treated with anti-CD19 chimeric antigen receptor (CAR)-T. Despite this clinical success, FDA-approved T-cell therapies are currently limited to B-cell malignancies, and challenges remain with managing cytokine-related toxicities. We have designed a novel antibody-T-cell receptor (AbTCR) platform where we combined the Fab domain of an antibody with the γ and δ chains of the TCR as the effector domain. We demonstrate the ability of anti-CD19-AbTCR-T cells to trigger antigen-specific cytokine production, degranulation, and killing of CD19-positive cancer cells in vitro and in xenograft mouse models. By using the same anti-CD19 binding moiety on an AbTCR compared to a CAR platform, we demonstrate that AbTCR activates cytotoxic T-cell responses with a similar dose-response as CD28/CD3ζ CAR, yet does so with less cytokine release and results in T cells with a less exhausted phenotype. Moreover, in comparative studies with the clinically validated CD137 (4-1BB)-based CAR, CTL019, our anti-CD19-AbTCR shows less cytokine release and comparable tumor inhibition in a patient-derived xenograft leukemia model.

Targeting Alpha-Fetoprotein (AFP)-MHC Complex with CAR T-Cell Therapy for Liver Cancer.

PURPOSE: The majority of tumor-specific antigens are intracellular and/or secreted and therefore inaccessible by conventional chimeric antigen receptor (CAR) T-cell therapy. Given that all intracellular/secreted proteins are processed into peptides and presented by class I MHC on the surface of tumor cells, we used alpha-fetoprotein (AFP), a specific liver cancer marker, as an example to determine whether peptide-MHC complexes can be targets for CAR T-cell therapy against solid tumors. EXPERIMENTAL DESIGN: We generated a fully human chimeric antigen receptor, ET1402L1-CAR (AFP-CAR), with exquisite selectivity and specificity for the AFP158-166 peptide complexed with human leukocyte antigen (HLA)-A*02:01. RESULTS: We report that T cells expressing AFP-CAR selectively degranulated, released cytokines, and lysed liver cancer cells that were HLA-A*02:01+/AFP+ while sparing cells from multiple tissue types that were negative for either expressed proteins. In vivo, intratumoral injection of AFP-CAR T cells significantly regressed both Hep G2 and AFP158-expressing SK-HEP-1 tumors in SCID-Beige mice (n = 8 for each). Moreover, intravenous administration of AFP-CAR T cells in Hep G2 tumor-bearing NSG mice lead to rapid and profound tumor growth inhibition (n = 6). Finally, in an established intraperitoneal liver cancer xenograft model, AFP-CAR T cells showed robust antitumor activity (n = 6). CONCLUSIONS: This study demonstrates that CAR T-cell immunotherapy targeting intracellular/secreted solid tumor antigens can elicit a potent antitumor response. Our approach expands the spectrum of antigens available for redirected T-cell therapy against solid malignancies and offers a promising new avenue for liver cancer immunotherapy. Clin Cancer Res; 23(2); 478-88. ©2016 AACR.

Potent T cell responses induced by single DNA vaccine boosted with recombinant vaccinia vaccine.

Plasmid DNA, an effective vaccine vector, can induce both cellular and humoral immune responses. However, plasmid DNA raises issues concerning potential genomic integration after injection. This issue should be considered in preclinical studies. Tiantan vaccinia virus (TV) has been most widely utilized in eradicating smallpox in China. This virus has also been considered as a successful vaccine vector against a few infectious diseases. Potent T cell responses through T-cell receptor (TCR) could be induced by three injections of the DNA prime vaccine followed by a single injection of recombinant vaccinia vaccine. To develop a safer immunization strategy, a single DNA prime followed by a single recombinant Tiantan vaccinia (rTV) AIDS vaccine was used to immunize mice. Our data demonstrated that one DNA prime/rTV boost regimen induced mature TCR activation with high functional avidity, preferential T cell Vß receptor usage and high sensitivity to anti-CD3 antibody stimulation. No differences in T cell responses were observed among one, two or three DNA prime/rTV boost regimens. This study shows that one DNA prime/rTV boost regimen is sufficient to induce potent T cell responses against HIV.

Nevirapine inhibits the anti-HIV activity of CD8+ cells.

Antiretroviral therapy (ART) significantly reduced the CD8 cell noncytotoxic anti-HIV response in 12 HIV-1-infected subjects (P < 0.0001). In separate experiments, CD8(+) cells from long-term survivors were cocultured with HIV-infected CD4(+) cells using varying concentrations of anti-HIV drugs. The antiviral function of CD8(+) cells from 4 of the 14 LTSs was reduced with exposure to 10 µM of nevirapine (P < 0.05). The antiviral activity of CD8(+) cells from 2 LTSs was inhibited by 5 µM of zidovudine. These studies indicate that nevirapine and probably zidovudine can inhibit the anti-HIV activity of CD8(+) cells and thus could influence the effectiveness of antiretroviral therapy.

Broad HIV-1 neutralizing antibody response induced by heterologous gp140/gp145 DNA prime-vaccinia boost immunization.

OBJECTIVE: To develop an effective HIV vaccine strategy that can induce cross-reactive neutralizing antibody. METHODS: Codon-optimized gp140 and gp145 env genes derived from HIV-1(cn54), a CRF07 B'/C recombinant strain, were constructed as DNA and recombinant Tiantan vaccinia (rTV) vaccines. The effect of heterologous immunization with gp140 and gp145 was tested in mice and guinea pigs. T cell responses were detected using the IFN-γ ELISPOT assay. A panel of primary isolates of clade B' and B'/C HIV-1 and TZM-bl cells was used to determine the neutralizing activity of immunized sera. RESULTS: The neutralizing antibodies (NAbs) induced by the heterologous immunogen immunization neutralized all HIV-1 B' and B'/C primary isolates in the guinea pig model. Gp145 and gp140 heterologous prime-boost induced the best neutralizing antibody response with a broad neutralizing spectrum and the highest titer of 1:270 at 6 weeks after the last inoculation. However, the T cell response to HIV-1 peptides was significantly weaker than the gp145+gp145 homologous prime-boost. CONCLUSIONS: This heterologous prime-boost immunization strategy could be used to design immunogen-generating broad neutralizing antibodies against genetic variance pathogens.

Transfection optimization for primary human CD8+ cells.

Electroporation, a non-virus-mediated gene transfection method, has traditionally had poor outcomes with low gene transfection efficiency and poor cellular viability, particularly in primary human lymphocytes. Herein we have optimized the electroporation conditions for primary CD8+ cells resulting in a maximum rate of 81.3%, and a mean transfection efficiency of 59.6%. After removal of dead cells, the viability of transfected primary CD8+ cells was greater than 90%, similar to untransfected controls. Using this procedure, primary human CD8+ cells transfected with an interferon α8 plasmid produced fluids that inhibited HIV-1 replication by > 95%. This transfection protocol is useful for transfection of other primary blood cells, such as CD4+ T cells, and for studying the function of genes in primary human blood cells instead of cell lines. The transfection procedure also has potential application in gene therapy clinical trials to treat diseases utilizing transfected primary human cells.

Broader HIV-1 neutralizing antibody responses induced by envelope glycoprotein mutants based on the EIAV attenuated vaccine.

BACKGROUND: In order to induce a potent and cross-reactive neutralizing antibody (nAb), an effective envelope immunogen is crucial for many viral vaccines, including the vaccine for the human immunodeficiency virus (HIV). The Chinese equine infectious anemia virus (EIAV) attenuated vaccine has controlled the epidemic of this virus after its vaccination in over 70 million equine animals during the last 3 decades in China. Data from our past studies demonstrate that the Env protein of this vaccine plays a pivotal role in protecting horses from both homologous and heterogeneous EIAV challenges. Therefore, the amino acid sequence information from the Chinese EIAV attenuated vaccine, in comparison with the parental wild-type EIAV strains, was applied to modify the corresponding region of the envelope glycoprotein of HIV-1 CN54. The direction of the mutations was made towards the amino acids conserved in the two EIAV vaccine strains, distinguishing them from the two wild-type strains. The purpose of the modification was to enhance the immunogenicity of the HIV Env. RESULTS: The induced nAb by the modified HIV Env neutralized HIV-1 B and B'/C viruses at the highest titer of 1:270. Further studies showed that a single amino acid change in the C1 region accounts for the substantial enhancement in induction of anti-HIV-1 neutralizing antibodies. CONCLUSIONS: This study shows that an HIV envelope modified by the information of another lentivirus vaccine induces effective broadly neutralizing antibodies. A single amino acid mutation was found to increase the immunogenicity of the HIV Env.

A mouse model based on replication-competent Tiantan vaccinia expressing luciferase/HIV-1 Gag fusion protein for the evaluation of protective efficacy of HIV vaccine.

BACKGROUND: Developing an effective vaccine against human immunodeficiency virus type 1 (HIV-1) remains a grand challenge after more than two decades of intensive effort. It is partially due to the lack of suitable animal models for screening and prioritizing vaccine candidates. In this study, we aim to develop a mice model to test HIV-1 vaccine efficacy. METHODS: We constructed a recombinant vaccinia expressing firefly luciferase and HIV-1 Gag fusion protein based on Tiantan strain, an attenuated but replication-competent poxvirus (rTTV-lucgag). By quantifying the luciferase activity as its read out, we defined the biodistribution of Tiantan strain poxvirus in mice inoculated intraperitoneally and attempted to apply this model to evaluate the HIV-1 vaccine efficacy. RESULTS: Our data demonstrated that the rTTV-lucgag was able to express high level of luciferase (< or = 10(6) relative luciferase units (RLU)/mg protein) and HIV-1 Gag (> 3 folds increase comparing to the control). After intraperitoneal inoculation, this virus had dominant replication in the ovary, uterus, and cervix of mice and the luciferase activities in those organs are significantly correlated with viral titers (r(2) = 0.71, P < 0.01). Pre-immunization with an HIV gag DNA vaccine reduced the luciferase activity in ovary from (6006 +/- 3141) RLU/mg protein in control group to (1538 +/- 463) RLU/mg protein in vaccine group (P = 0.1969). CONCLUSIONS: The luciferase activity in ovary could represent viral replication in vivo; this rTTV-lucgag/mice model may be suitable to assess the protective efficacy of cytotoxic T-cell responses to HIV Gag with less tedious work and high through-put.

Deglycosylation or partial removal of HIV-1 CN54 gp140 V1/V2 domain enhances env-specific T cells.

It remains a great challenge to develop an effective HIV vaccine against the most prevalent HIV-1 clade, B'/C recombinant, in China. Our objective was to test the influence of a new modification of the V1/V2 loops of HIV-1(CN54) gp140 on the immunogenicity of Env. HIV-1(CN54) gp140 was deglycosylated by replacing all six N residues in V1/V2 loops with six Q residues (gp140dG) or partially deleted on V1/V2 loops (gp140dV). gp140, gp140dG, and gp140dV were transferred into plasmid vector and recombinant Tiantan vaccinia (rTTV) vector to generate three DNA vaccines and three rTTV vaccines for vaccination of female BALB/c mice in a prime-boost regimen. An Elispot assay was used to read out the T cell immunity and ELISA and a poly-l-leucine (PLL) ELISA was employed to assess humoral immune responses. Surprisingly, gp140dV (1570 +/- 1569 SFCs/10(6) splenocytes) and gp140dG (731 +/- 471 SFCs/10(6) splenocytes) could elicit significantly higher Env-specific T cells than gp140 (224 +/- 140 SFCs/10(6) splenocytes). Three T cell epitopes were newly identified in BALB/c mice at the N terminus of C1, C terminus of C4, and N terminus of HR, respectively. Env-specific binding antibodies and linear antibodies elicited by gp140 tended to be higher than that stimulated by gp140dG and gp140dV but did not reach statistical difference. Our data demonstrated that the deglycosylation and partial deletion of V1/V2 loops of B'/C recombinant gp140 could lead to improvement of specific T cell immune responses.

Pathogenicity and immunogenicity of recombinant Tiantan Vaccinia Virus with deleted C12L and A53R genes.

Interest is increasing regarding replicating poxvirus as HIV vaccine vector. In China, the Tiantan Vaccinia Virus (TV) has been used most extensively in the battle of eradicating smallpox. Recently, TV was developing as vaccine vector to fight against infectious diseases such as human immunodeficiency virus (HIV). However, replicating vaccinia virus sometimes may pose serious post-vaccination complications, especially in immunosuppressed individuals. To develop a safer and more effective TV-based vector, we constructed C12L (vIL-18 binding protein) and A53R (vTNF receptor homolog) gene-deleted mutants which are based on parental TV and VTKgpe (TV expressing HIV gagpol and env gene), respectively. The pathogenicity and immunogenicity were also evaluated. Deleting these two immunomodulatory genes lessened the virulence of the parental virus in both mice and rabbit models. Notably, C12L deletion mutant attenuated the skin virulence of parental virus by as high as approximate 2 logs. Furthermore, VTKgpe with A53R and C12L gene deletion retains the high immunogenicity of the parental virus to elicit strong humoral and cellular responses to the HIV target genes despite the remarkable attenuation. These data suggest that deletion of the cytokine viroceptor gene is feasible to obtain a safer and replication-competent TV vector for vaccination and immunotherapy.

[Effect of GBV-C/HIV coinfection on HIV/AIDS disease progression and HIV replication].

Several research groups have recently reported that persistent GB virus C (GBV-C) co-infected with human immunodeficiency virus (HIV) leads to slower AIDSs disease progression than HIV-1 infection alone. However, these findings were not confirmed by several other studies. To investigate the association between GBV-C replication and plasma HIV loads and CD4+ T cell counts, 203 HIV-1 positive former blood/plasma donors(FBDs) were enrolled from Fuyang city of Anhui Province in China. Plasma specimens were collected from them and were tested for GBV-C using RT-PCR and ELISA. Out of 203 specimens, 52 (25.6%) cases were positive for GBV-C, including 35 male (67.3%) and 17 female (32.7%) cases. No significant association was identified between GBV-C infection and CD4+ T-cell counts or between GBV-C infection and HIV viral loads. Since all the subjects studied were naive to ART, the influence of therapy on AIDS disease progression was ruled out in this study. Overall, our data indicated that HIV-1 positive male FBDs were prone to be infected, GBV-C coinfection with HIV-1 does not significantly influence HIV/AIDS disease progression during the late stage of chronic HIV-1 infection.

Truncation of cytoplasmic tail of EIAV Env increases the pathogenic necrosis.

Equine Infectious Anemia Virus (EIAV), like other lentiviruses, has a transmembrane glycoprotein with an unusually long cytoplasmic tail (CT). Viral envelope (Env) proteins having CT truncations just downstream the putative membrane-spanning domain (PMSD) are assumed to exist among all wild-type budded virions, and also in some cell-adapted strains. To determine whether CT-truncated Env proteins can cause particularly deleterious effects on the Env expressing cells and/or their neighboring cells, plasmids encoding codon-optimized env gene including full-length (pE863) or CT-truncated (pE686* and pE676*) were transiently transfected into 293T cells, respectively. Data from intracellular protein expression and cell death assays revealed that CT-truncated Env, compared to full-length Env, not only induced comparable apoptosis, but also caused much more intensive mitochondria-mediated necrosis that could simultaneously induce significant decrease of intracellular protein expression in the Env expressing cells. Moreover, results from flow cytometric analysis showed that mitochondrial depolarization preceded the caspase activation in cells no matter which env construct was delivered, and indicated that both full-length and CT-truncated Env proteins share a common intrinsic mitochondrial pathway to induce apoptosis. Our results partially elucidate the mechanisms underlying cell death resulting from EIAV pathogenesis.

Mucosal priming with replicative Tiantan vaccinia and systemic boosting with DNA vaccine raised strong mucosal and systemic HIV-specific immune responses.

An effective vaccine strategy for HIV-1 will probably require the induction and maintenance of both humoral and cellular immunity at mucosal and systemic sites. We tested a new prime-boost approach of intranasal priming with 3 x 10(6) PFU of replicative recombinant Tiantan vaccinia virus (rTTV) and intramuscular boosting with 100 microg DNA plasmid expressing HIV-1 Gag in BALB/c mice along with other strategies. Our data demonstrated that intranasal priming with replicative recombinant Tiantan vaccinia and intramuscular boosting with DNA vaccine raised the highest vaginal IgA and systemic T-cell responses, and modest lung IgA and sera IgG responses among all vaccination regimens; each vaccination regimen generated its own imprint of the most preferential T-cell receptor usage of Vbeta. These results demonstrate that the combination of intranasal priming with replicative recombinant Tiantan vaccinia and intramuscular boosting with DNA vaccine is a preferable regimen for induction of both T-cell and humoral immune responses at mucosal and systemic sites.

Comparison of immunogenicity between codon optimized HIV-1 Thailand subtype B gp140 and gp145 vaccines.

HIV-1 pandemic posed an unprecedented challenge to the global health and it is believed that an effective vaccine will be the final solution against HIV-1. HIV-1 envelope is the primary immunogen in developing neutralization antibody based HIV vaccine. To define the suitable Env derived immunogen, we systemically compared the immunogenicity of gp140 and gp145 in a DNA vaccination alone and a prime-boost modalities. Two DNA vaccines and two recombinant Tiantan vaccinia vaccines (rTTV) were constructed for vaccination of female Balb/c mice. Elispot assay was used to read out the T cell immunity and ELISA assay was used to quantify antibody immunity. PLL (poly-L-leucine)-ELISA assay was used in linear antibody epitope mapping. Mice primed with gp145 tended to elicit more Env-specific T cells responses than those primed with gp140, significant difference was observed in DNA immunization alone. The ultimate T cell responses in prime-boost regimen tend to be determined mainly by the priming efficacy. Linear antibody epitope mapping displayed that sera raised by gp145 priming were vigorously reactive to more peptides than that by gp140. Our data demonstrated HIV-1 Thailand B-derived gp145 may raise higher T-cell responses and broader linear peptide-specific antibody responses than gp140 does. However, it remains to be determined that how these observations are relevant to the neutralization of antibody activities.

Mucosal priming with PEI/DNA complex and systemic boosting with recombinant TianTan vaccinia stimulate vigorous mucosal and systemic immune responses.

An effective vaccine strategy for HIV-1 will probably requires the induction and maintenance of both humoral and cellular immunity. We tested a new prime-boost approach of intranasal priming with 10 microg DNA plasmid in the PEI/DNA complexes and boosting with 10(7)PFU of replicative recombinant TianTan vaccinia virus (rTTV) expressing HIV-1 Gag in BALB/c mice. Intranasal priming with PEI/DNA complexes elicited strikingly stronger HIV-specific T-cell (p=0.0358) and IgA immune responses at mucosal sites of lung (p=0.0445) and vaginal tract (p=0.0469) than intranasal priming with naked DNA, though both are followed by the same rTTV boosting. Furthermore, an intramuscular boosting with rTTV could profoundly enhance both T-cell and antibody immune responses raised by intranasal priming. These results demonstrate that the combination of intranasal priming with PEI/DNA complexes and systemic boosting with rTTV is a preferable regimen for induction of both T-cell and humoral immune responses.

Immunogenicity comparison between codon optimized HIV-1 CRF BC_07 gp140 and gp145 vaccines.

To develop an effective vaccine against the most prevalent HIV strain "B'/C recombinant" in China, we compared the immunogenicity of B'/C-derived gp140 and gp145. The codon optimized gp140 and gp145 env gene derived from CN54, an ancestor-like B'/C recombinant strain, were synthesized and cloned into a plasmid as DNA vaccines, designated as pDRVISV140 and pDRVISV145, respectively. BALB/c mice were inoculated three times at week 0, 2, and 4 and sacrificed at week 7. Both T cell immunity and humoral immunity were determined. The mock vector pDRVISV1.0 carrying no HIV immunogen was included as control. Our data showed that B'/C recombinant-derived gp145 mounted stronger T cell and broader linear antibody but less binding antibody immune responses than gp140 did. Though both gp145 and gp140 raised neutralization antibodies against laboratory-adapted strain SF33, both failed to neutralize B' or B'/C clade primary strains. Overall, this is the first time the immunogenicity of B'/C recombinant-derived gp140 and gp145 was examined and compared; our data prefer B'/C-derived gp145 to gp140 as an HIV vaccine immunogen. The failure to induce neutralization antibodies against primary isolates indicates that it is insufficient to enhance the immunogenicity of conserved epitopes by simply employing gp145 or gp140; strategies to enhance antibody responses against conserved epitopes should be explored further.

[Intranasal priming with HIV DNA vaccine and systemic boosting with recombinant vaccinia induce vigorous immune responses: experiment with mice].

OBJECTIVE: To explore the strategy to raise both mucosal and systemic anti-HIV-1 immunity. METHODS: Eighteen BALB/c rats were randomly divided into 2 groups, experimental group and control group. The experimental group were further subdivided into 4 subgroups of 3 mice: 3-dose HIV DNA vaccine group, 3-dose DNA vaccine + cholera toxin (CT) adjuvant subgroup, 1-dose recombinant Tiantan strain vaccinia-based vaccine subgroup, and 3-dose DNA vaccine + CT adjuvant + Tiantan strain vaccinia-based vaccine subgroup. The control group was subdivided into 2 subgroups of 3 mice: 3-dose DNA blank vector subgroup, and 3-dose DNA blank vector + Tiantan strain vaccinia-based vaccine subgroup. Intranasal administration of DNA vaccine-based vaccine (10 microg) was done on the days 0, 14, and 28 as the mucosal priming, and recombinant Tiantan vaccinia (1 x 10(7) PFU) was injected intramuscularly as systemic boosting on the day 42. On the day 56 the mice were killed and specimens of serum, nasopharynx wash, lung wash, and spleen were collected and splenocytes were isolated. Splenocytes were added into the phosphate-buffered saline with anti-mouse interferon-gamma (IFN-gamma) envelop antibody to count the number of spot-forming cells (SFCs). Indirect ELISA was used to detect the HIV-1 specific antibody in the nasopharynx wash and lung wash. Immunohistochemistry was used to detect the intracellular staining of IFN-gamma in the splenocytes. RESULTS: The number of spot forming cells in the HIV-1 DNA vaccine + CT adjuvant group was (14 +/- 11) SFCs/10(6) splenocytes, significantly more than that of the HIV-1 DNA vaccine group [(2 +/- 1) SFCs/10(6) spleen cells (P < 0.01). The number of SFCs of the 1-dose DNA-vaccine subgroup was [(30 +/- 18) SFCs/10(6) spleen cells], significantly higher than that of the only DNS vaccine group (P < 0.01). The number of SFCs of DNA vaccine + CT adjuvant + recombinant Tiantan vaccinia-based vaccine was (61 +/- 35) SFCs/10(6) splenocytes, significantly higher than those of the other groups (all P < 0.01). Flow cytometry showed that the rate of HIV-1 Gag specific CD8(+) T cell was 1.8% +/- 1.4%. The value of specific IgG of the DNA vaccine + adjuvant + Tiantan vaccinia-based vaccine was 1.50 +/- 0.30, significantly higher than those of the blank vector, single-dose Tiantan vaccinia-based vaccine, and single-dose DNA vaccine + CT adjuvant subgroups (0.42 +/- 0.02, 0.74 +/- 0.13, and 0.75 +/- 0.02 respectively, all P < 0.05). In different subgroups the levels of specific IgA in the lung wash were all higher than those in the nasopharynx wash. The levels of specific IgA in the lung and nasopharynx wash of the DNA vaccine + CT adjuvant subgroup were higher than those of the other subgroups whether or not with boosting of Tiantan. The specific IgA levels of the groups enhanced by Tiantan vaccinia-based vaccine were all significantly higher than those of the corresponding subgroups without enhancement (all P < 0.01). The IgA level of lung wash of the DNA vaccine + CT adjuvant subgroup was 1.82 +/- 0.76, significantly higher than that of the one-dose Tiantan vaccinia-based vaccine group (0.52 +/- 0.19, P < 0.05). CONCLUSION: The vaccination modality of mucosal priming and systemic boosting induces both mucosal and systemic immune responses.