Protective immune response against cutaneous leishmaniasis by prime/booster immunization regimens with vaccinia virus recombinants expressing Leishmania infantum p36/LACK and IL-12 in combination with purified p36

In susceptible mice Leishmania infection triggers a CD4(+) Th2 response that has been correlated with evasion of the host immune system. To develop approaches that might trigger a Th1 response leading to protection against Leishmania we generated vaccinia virus recombinants (VVr) expressing the relevant p36/LACK protein of Leishmania infantum (VVp36) or co-expressing p36/LACK and interleukin-12 (VVp36IL12). Susceptible BALB/c mice were immunized with the VVr in various prime/booster protocols that included purified p36/LACK protein, followed 3 weeks later by a challenge with live L. major promastigotes. The course of the infection was monitored by measuring lesion development, parasite load and immunological parameters (IFN-gamma and IL-10 secretion by in vitro-stimulated lymphocytes, and specific IgG isotypes), before and after challenge. We found protocols of prime/booster immunization (VVp36/VVp36; VVp36IL12/p36; p36/VVp36IL12) that elicited different levels of protection in infected animals. The protocol of priming with purified p36 followed by a booster with VVp36IL12 induced 52% reduction in lesion size and a two-log unit reduction in parasite load. This partial protection correlated with activation of a specific Th1 type of immune response. These protocols could be of interest in the prophylaxis against Leishmania spp. and other parasitic diseases.     

Molecular cloning, cell localization and binding affinity to DNA replication proteins of the p36/LACK protective antigen from Leishmania infantum.

The p36/LACK antigen from Leishmania, an analogue of the receptor for activated protein kinase C (PKC), induces high levels of protection against parasite infection in the BALB/c mouse model. This protection is more than twice as high as that elicited by major parasite antigens such as soluble Leishmania antigen or the main surface protease gp63. We have cloned and purified p36/LACK from Leishmania infantum, the causative agent of visceral leishmaniasis in Europe. This protein belongs to the large family of WD 40 repeat proteins confined to eukaryotes and involved in numerous regulatory functions. Differential solubilization and immunofluorescence experiments indicate that p36/LACK is present close to the kinetoplast disc in the cell cytoplasm, probably bound to multiprotein complexes but not to membrane structures. These complexes probably also include cytoplasm PKC isoforms. The use of a genetically-encoded peptide library indicates that p36/LACK binds sequences present in several proteins involved in DNA replication and RNA synthesis. The recognition and binding sequences present in vacuolar proteins and at the beta-chain of major histocompatability complex (MHC) class II suggest the involvement of this regulatory protein in the early mechanisms triggering the protective immune response of the host against the parasite infection.     

MVA-LACK as a safe and efficient vector for vaccination against leishmaniasis

An optimal vaccine against leishmaniasis should elicit parasite specific CD4+ and cytotoxic CD8+ T cells. In this investigation, we described a prime/boost immunization approach based on DNA and on poxvirus vectors (Western Reserve, WR, and the highly attenuated modified vaccinia virus Ankara, MVA), both expressing the LACK antigen of Leishmania infantum, that triggers different levels of specific CD8+ T cell responses and protection (reduction in lesion size and parasitemia) against L. major infection in mice. A prime/boost vaccination with DNA-LACK/MVA-LACK elicits higher CD8+ T cell responses than a similar protocol with the replication competent VV-LACK. Both CD4+ and CD8+ T cells were induced by DNA-LACK/MVA-LACK immunization. The levels of IFN-gamma and TNF-alpha secreting CD8+ T cells were higher in splenocytes from DNA-LACK/MVA-LACK than in DNA-LACK/VV-LACK immunized animals. Moreover, protection against L. major was significantly higher in DNA-LACK/MVA-LACK than in DNA-LACK/VV-LACK immunized animals when boosted with the same virus dose, and correlated with high levels of IFN-gamma and TNF-alpha secreting CD8+ T cells. In DNA-LACK/MVA-LACK vaccinated animals, the extent of lesion size reduction ranged from 65 to 92% and this protection was maintained for at least 17 weeks after challenge with the parasite. These findings demonstrate that in heterologous prime/boost immunization approaches, the protocol DNA-LACK/MVA-LACK is superior to DNA-LACK/VV-LACK in triggering specific CD8+ T cell immune responses and in conferring protection against cutaneous leishmaniasis. Thus, MVA-LACK is a safe and efficient vector for vaccination against leishmaniasis.     

Interleukin-12 (IL-12) enhancement of the cellular immune response against human immunodeficiency virus type 1 env antigen in a DNA prime/vaccinia virus boost vaccine regimen is time and dose dependent: suppressive effects of IL-12 boost are mediated by nitric oxide

We previously demonstrated that codelivery of interleukin-12 (IL-12) with the human immunodeficiency virus type 1 (HIV-1) Env antigen from a recombinant vaccinia virus (rVV) can enhance the specific anti-Env cell-mediated immune (CMI) response. In the present study, we have investigated the effects of IL-12 in mice when it is expressed in a DNA prime/VV boost vaccine regimen. The delivery of IL-12 and Env product during priming with a DNA vector, followed by a booster with VV expressing the Env gene (rVVenv), was found to trigger the optimal CMI response compared with other immunization schedules studied. Significantly, if IL-12 is also delivered as a booster from the viral vector, an impairment of the effects of IL-12 was observed involving nitric oxide (NO), since it was overcome by specific inhibitors of inducible NO synthase. NO caused transient immunosuppression rather than impairment of viral replication. Moreover, at certain viral doses, coadministration of the NO inhibitor during the booster resulted in IL-12-mediated enhancement of the specific CD8(+) T-cell response. In addition, the dose of the IL-12-encoding plasmid (pIL-12) and the route of administration of both vectors were relevant factors for optimal CMI responses. Maximal numbers of Env-specific CD8(+) gamma interferon-secreting cells were obtained when 50 microg of pIL-12 was administered intramuscularly at priming, followed by an intravenous rVVenv boost. Our results demonstrate, in a murine model, critical parameters affecting the success of vaccination schedules based on a combination of DNA and VV vectors in conjunction with immunomodulators.     

High quality long-term CD4+ and CD8+ effector memory populations stimulated by DNA-LACK/MVA-LACK regimen in Leishmania major BALB/c model of infection

Heterologous vaccination based on priming with a plasmid DNA vector and boosting with an attenuated vaccinia virus MVA recombinant, with both vectors expressing the Leishmania infantum LACK antigen (DNA-LACK and MVA-LACK), has shown efficacy conferring protection in murine and canine models against cutaneus and visceral leishmaniasis, but the immune parameters of protection remain ill defined. Here we performed by flow cytometry an in depth analysis of the T cell populations induced in BALB/c mice during the vaccination protocol DNA-LACK/MVA-LACK, as well as after challenge with L. major parasites. In the adaptive response, there is a polyfunctional CD4(+) and CD8(+) T cell activation against LACK antigen. At the memory phase the heterologous vaccination induces high quality LACK-specific long-term CD4(+) and CD8(+) effector memory cells. After parasite challenge, there is a moderate boosting of LACK-specific CD4(+) and CD8(+) T cells. Anti-vector responses were largely CD8(+)-mediated. The immune parameters induced against LACK and triggered by the combined vaccination DNA/MVA protocol, like polyfunctionality of CD4(+) and CD8(+) T cells with an effector phenotype, could be relevant in protection against leishmaniasis.     

IBV S1基因和IL-2基因双顺反子DNA疫苗的免疫原性研究

为了研制更加有效的IBV DNA疫苗,将IBV的S1基因和禽白介素2(IL-2)基因插入双顺反子表达载体pIRES-EGFP/DsRed中,构建能分别或同时表达S1基因和IL-2基因的pIRES-S1、pIRES-IL2、pIRES-S1/IL-2质粒。通过脂质体转染Vero细胞,利用RT-PCR及间接免疫荧光检测表达。将构建的质粒用脂质体包裹后,通过腿部肌肉多点注射免疫7日龄雏鸡,二免后两周用IBV肾型强毒进行攻毒。结果表明,pIRES-S1/IL-2在体外能够诱导Vero细胞表达S1蛋白和IL-2;pIRES-S1/IL-2和pIRES-S1 pIRES-IL2免疫雏鸡后均能促进外周血T淋巴细胞亚群数量和血清中特异性抗体水平的增加,能明显增强IBV DNA疫苗对同型强毒的攻击保护,但pIRES-S1/IL-2免疫组要优于pIRES-S1 pIRES-IL2混合免疫组及其它对照组,差异显著或极显著。以上结果表明禽IL-2能同时加强DNA疫苗的细胞免疫和体液免疫应答;但抗原基因和IL-2共表达DNA疫苗的免疫效果明显要优于混合注射的DNA疫苗。     

Immunization against Leishmania major infection using LACK- and IL-12-expressing Lactococcus lactis induces delay in footpad swelling

Background

Leishmania is a mammalian parasite affecting over 12 million individuals worldwide. Current treatments are expensive, cause severe side effects, and emerging drug resistance has been reported. Vaccination is the most cost-effective means to control infectious disease but currently there is no vaccine available against Leishmaniasis. Lactococcus lactis is a non-pathogenic, non-colonizing Gram-positive lactic acid bacterium commonly used in the dairy industry. Recently, L. lactis was used to express biologically active molecules including vaccine antigens and cytokines.

Methodology/Principal findings

We report the generation of L. lactis strains expressing the protective Leishmania antigen, LACK, in the cytoplasm, secreted or anchored to the bacterial cell wall. L. lactis was also engineered to secrete biologically active single chain mouse IL-12. Subcutaneous immunization with live L. lactis expressing LACK anchored to the cell wall and L. lactis secreting IL-12 significantly delayed footpad swelling in Leishmania major infected BALB/c mice. The delay in footpad swelling correlated with a significant reduction of parasite burden in immunized animals compared to control groups. Immunization with these two L. lactis strains induced antigen-specific multifunctional T_H1 CD4⁺ and CD8⁺ T cells and a systemic LACK-specific T_H1 immune response. Further, protection in immunized animals correlated with a Leishmania-specific T_H1 immune response post-challenge. L. lactis secreting mouse IL-12 was essential for directing immune responses to LACK towards a protective T_H1 response.

Conclusions/Significance

This report demonstrates the use of L. lactis as a live vaccine against L. major infection in BALB/c mice. The strains generated in this study provide the basis for the development of an inexpensive and safe vaccine against the human parasite Leishmania.     

Immunization with a vaccinia virus recombinant expressing herpes simplex virus type 1 glycoprotein D: long-term protection and effect of revaccination.

Previously we showed that mice immunized with a vaccinia virus vector expressing the herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) gene (vaccinia/gD) were protected against both lethal and latent infections with HSV-1 for at least 6 weeks after immunization (K. J. Cremer, M. Mackett, C. Wohlenberg, A. L. Notkins, and B. Moss, Science 228:737-740, 1985). In the experiments described here, we examined long-term immunity to HSV following vaccinia/gD vaccination, the effect of revaccination with vaccinia/gD, and the impact of previous immunity to vaccinia virus on immunization with the gD recombinant. Mice immunized with vaccinia/gD showed 100, 100, and 80% protection against lethal infection with HSV-1 at 18, 44, and 60 weeks postimmunization, respectively. Protection against latent trigeminal ganglionic infection was 70, 50, and 31% at 6, 41, and 60 weeks postvaccination, respectively. To study the effect of reimmunization on antibody levels, mice vaccinated with vaccinia/gD were given a second immunization (booster dose) 3 months after the first. These mice developed a 10-fold increase in neutralizing-antibody titer (221 to 2,934) and demonstrated a significant increase in protection against lethal HSV-1 challenge compared with animals that received only one dose of vaccinia/gD. To determine whether preexisting immunity to vaccinia virus inhibited the response to vaccination with vaccinia/gD virus, mice were immunized with a recombinant vaccinia virus vector expressing antigens from either influenza A or hepatitis B virus and were then immunized (2 to 3 months later) with vaccinia/gD. These mice showed reduced titers of neutralizing antibody to HSV-1 and decreased protection against both lethal and latent infections with HSV-1 compared with animals vaccinated only with vaccinia/gD. We conclude that vaccination with vaccinia/gD produces immunity against HSV-1 that lasts over 1 year and that this immunity can be increased by a booster but that prior immunization with a vaccinia recombinant virus expressing a non-HSV gene reduces the levels of neutralizing antibody and protective immunity against HSV-1 challenge.     

Requirements for the maintenance of Th1 immunity in vivo following DNA vaccination: a potential immunoregulatory role for CD8+ T cells

Protective immunity against Leishmania major generated by DNA encoding the LACK (Leishmania homologue of receptor for activated C kinase) Ag has been shown to be more durable than vaccination with LACK protein plus IL-12. One mechanism to account for this may be the selective ability of DNA vaccination to induce CD8+ IFN-gamma-producing T cells. In this regard, we previously reported that depletion of CD8+ T cells in LACK DNA-vaccinated mice abrogated protection when infectious challenge was done 2 wk postvaccination. In this study, we extend these findings to study the mechanism by which CD8+ T cells induced by LACK DNA vaccination mediate both short- and long-term protective immunity against L. major. Mice vaccinated with LACK DNA and depleted of CD8+ T cells at the time of vaccination or infection were unable to control infection when challenge was done 2 or 12 wk postvaccination. Remarkably, it was noted that depletion of CD8+ T cells in LACK DNA-vaccinated mice was associated with a striking decrease in the frequency of LACK-specific CD4+ IFN-gamma-producing T cells both before and after infection. Moreover, data are presented to suggest a mechanism by which CD8+ T cells exert this regulatory role. Taken together, these data provide additional insight into how Th1 cells are generated and sustained in vivo and suggest a potentially novel immunoregulatory role for CD8+ T cells following DNA vaccination.     

Towards a new generation of vaccines: the cytokine IL-12 as an adjuvant to enhance cellular immune responses to pathogens during prime-booster vaccination regimens

A main goal of the industrialized world is the development of effective vaccines to control infectious diseases with major health and socio-economic impact. Current understanding of the immune response triggered during infection with pathogens causing malaria, hepatitis C and AIDS emphasizes the importance of cytotoxic T lymphocytes (CTLs) in combating these infections. This has led to the development of new vaccination strategies, some of which are in phase I/II clinical trials. Promising strategies of vaccination are based on highly attenuated viral vectors, such as Vaccinia virus (VV) in combination with heterologous like vectors naked DNA, referred to as priming/booster vaccination. While these immunization schedules increased the production of specific CTLs, there is a need to further expand the CD8+T cell population to control an infection. Among molecules that play a significant role in the modulation of the CTL response is the cytokine IL-12. Immunoregulation by IL-12 is of central importance in cell-mediated immunity (CMI) against those pathogens and tumors that are controlled by cell-mediated mechanisms, supported by Thl cells. The use of this cytokine in combination with highly immunogenic VV-derived vectors is a promising system for development of future vaccination schedules. In this review, we summarize recent data on the use of IL-12 in vaccination procedures, as well as undesired side-effects of the cytokine that can be overcome by accurate use of dose, route and time-window administration of IL-12 encoding vectors. Results described here indicate that VV IL-12-mediated enhancement of the specific CMI response against a model antigen HIV-1 env was time- and dose-dependent and that the antigen and the cytokine could be expresed from two different rVVs modulating the doses of the vectors and allowing for enhancement of a specific CMI response. Moreover, the use of IL-12 during DNA prime/VV boost regimens enhanced the specific anti-HIV-1 env cellular response 20 times compared to that generated after a single rVVenv inoculation. Variables such as: a) dose of the cytokine applied, b) time of its administration and c) routes of inoculation play a critical role in the final outcome of the response. The findings presented here can be extended to other antigens, suggesting that immunomodulatory cytokines can be useful in the development of the future vaccines against numerous infectious diseases and tumors.     

Co-administration of plasmid expressing IL-12 with 14-kDa Schistosoma mansoni fatty acid-binding protein cDNA alters immune response profiles and fails to enhance protection induced by Sm14 DNA vaccine alone

Schistosomiasis is an endemic disease that affects 200 million people worldwide. DNA-based vaccine is a promising strategy to induce protective immunity against schistosomiasis, since both humoral and cellular immune responses are involved in parasite elimination. In this study, we evaluated the ability of Sm14 cDNA alone or in association with a plasmid expressing murine interleukin (IL)-12 to induce protection against challenge infection. Mice were immunized with four doses of the DNA vaccine and the levels of protection were determined by worm burden recovery after challenge infection. Specific antibody production to rSm14 was determined by ELISA, and cytokine production was measured in splenocyte culture supernatants stimulated with rSm14 and in bronchoalveolar lavage of vaccinated mice after challenge infection. DNA immunization with pCI/Sm14 alone induced 40.5% of worm reduction. However, the use of pCI/IL-12 as adjuvant to pCI/Sm14 immunization failed to enhance protection against challenge infection. Protection induced by pCI/Sm14 immunization correlates with specific IgG antibody production against Sm14, Th1 type of immune response with high levels of interferon (IFN)-gamma and low levels of IL-4 in splenocyte culture supernatants and in bronchoalveolar lavage after challenge infection. IL-12 co-administration with pCI/Sm14 induced a significant production of nitric oxide in splenocyte culture supernatants and also lymphocyte suppression, with reduced percentage of T cells producing IFN-gamma and tumor necrosis factor-alpha.     

Feline leukemia virus DNA vaccine efficacy is enhanced by coadministration with interleukin-12 (IL-12) and IL-18 expression vectors

The expectation that cell-mediated immunity is important in the control of feline leukemia virus (FeLV) infection led us to test a DNA vaccine administered alone or with cytokines that favored the development of a Th1 immune response. The vaccine consisted of two plasmids, one expressing the gag/pol genes and the other expressing the env gene of FeLV-A/Glasgow-1. The genetic adjuvants were plasmids encoding the feline cytokines interleukin-12 (IL-12), IL-18, or gamma interferon (IFN-gamma). Kittens were immunized by three intramuscular inoculations of the FeLV DNA vaccine alone or in combination with plasmids expressing IFN-gamma, IL-12, or both IL-12 and IL-18. Control kittens were inoculated with empty plasmid. Following immunization, anti-FeLV antibodies were not detected in any kitten. Three weeks after the final immunization, the kittens were challenged by the intraperitoneal inoculation of FeLV-A/Glasgow-1 and were then monitored for a further 15 weeks for the presence of virus in plasma and, at the end of the trial, for latent virus in bone marrow. The vaccine consisting of FeLV DNA with the IL-12 and IL-18 genes conferred significant immunity, protecting completely against transient and persistent viremia, and in five of six kittens protecting against latent infection. None of the other vaccines provided significant protection.     

SIR2-deficient Leishmania infantum induces a defined IFN-gamma/IL-10 pattern that correlates with protection

The ability to manipulate the Leishmania genome to create genetically modified parasites by introducing or eliminating genes is considered a powerful alternative for developing a new generation vaccine against leishmaniasis. Previously, we showed that the deletion of one allele of the Leishmania infantum silent information regulatory 2 (LiSIR2) locus was sufficient to dramatically affect amastigote axenic proliferation. Furthermore, LiSIR2 single knockout (LiSIR2(+/-)) amastigotes were unable to replicate in vitro inside macrophages. Because this L. infantum mutant persisted in BALB/c mice for up to 6 wk but failed to establish an infection, we tested its ability to provide protection toward a virulent L. infantum challenge. Strikingly, vaccination with a single i.p. injection of LiSIR2(+/-) single knockout elicits complete protection. Thus, vaccinated BALB/c mice showed a reversal of T cell anergy with specific anti-Leishmania cytotoxic activity and high levels of NO production. Moreover, vaccinated mice simultaneously generated specific anti-Leishmania IgG Ab subclasses suggestive of both type 1 and type 2 responses. A strong correlation was found between the elimination of the parasites and an increased Leishmania-specific IFN-gamma/IL-10 ratio. Therefore, we propose that the polarization to a high IFN-gamma/low IL-10 ratio after challenge is a clear indicator of vaccine success. Furthermore these mutants, which presented attenuated virulence, represent a good model to understand the correlatives of protection in visceral leishmaniasis.     

Intranasal immunization with chitosan microparticles enhances LACK-DNA vaccine protection and induces specific long-lasting immunity against visceral leishmaniasis

Development of a protective vaccine against Leishmania depends on antigen formulation and adjuvants that induce specific immunity and long-lasting immune responses. We previously demonstrated that BALB/c mice intranasally vaccinated with a plasmid DNA encoding the p36/LACK leishmanial antigen (LACK-DNA) develop a protective immunity for up to 3 months after vaccination, which was linked with the systemic expression of vaccine mRNA in peripheral organs. In this study, LACK-DNA vaccine was associated with biocompatible chitosan microparticles cross-linked with glyceraldehyde (CMC) to boost the long-lasting immunity against the late Leishmania infantum challenge. Infection at 7 days, 3 or 6 months after vaccination resulted in significantly lower parasite loads when compared with non-vaccinated controls. Besides, LACK-DNA-chitosan vaccinated mice showed long-time protection observed after the late time point challenge. The achieved protection was correlated with an enhanced spleen cell responsiveness to parasite antigens, marked by increased proliferation and IFN-γ as well as decreased IL-10 production. Moreover, we found diminished systemic levels of TNF-α that was compatible with the better health condition observed in LACK-DNA/CMC vaccinated-infected mice. Together, our data indicate the feasibility of chitosan microparticles as a delivery system tool to extend the protective immunity conferred by LACK-DNA vaccine, which may be explored in vaccine formulations against Leishmania parasite infections.     

Interleukin-23 restores immunity to Mycobacterium tuberculosis infection in IL-12p40-deficient mice and is not required for the development of IL-17-secreting T cell responses

Host control of Mycobacterium tuberculosis is dependent on the activation of CD4+ T cells secreting IFN-gamma and their recruitment to the site of infection. The development of more efficient vaccines against tuberculosis requires detailed understanding of the induction and maintenance of T cell immunity. Cytokines important for the development of cell-mediated immunity include IL-12 and IL-23, which share the p40 subunit and the IL-12Rbeta1 signaling chain. To explore the differential effect of IL-12 and IL-23 during M. tuberculosis infection, we used plasmids expressing IL-23 (p2AIL-23) or IL-12 (p2AIL-12) alone in dendritic cells or macrophages from IL-12p40(-/-) mice. In the absence of the IL-12/IL-23 axis, immunization with a DNA vaccine expressing the M. tuberculosis Ag85B induced a limited Ag-specific T cell response and no control of M. tuberculosis infection. Co-delivery of p2AIL-23 or p2AIL-12 with DNA85B induced strong proliferative and IFN-gamma-secreting T cell responses equivalent to those observed in wild-type mice immunized with DNA85B. This response resulted in partial protection against aerosol M. tuberculosis; however, the protective effect was less than in wild-type mice owing to the requirement for IL-12 or IL-23 for the optimal expansion of IFN-gamma-secreting T cells. Interestingly, bacillus Calmette-Guérin immune T cells generated in the absence of IL-12 or IL-23 were deficient in IFN-gamma production, but exhibited a robust IL-17 secretion associated with a degree of protection against pulmonary infection. Therefore, exogenous IL-23 can complement IL-12 deficiency for the initial expansion of Ag-specific T cells and is not essential for the development of potentially protective IL-17-secreting T cells.     

Rapid IL-4 production by Leishmania homolog of mammalian RACK1-reactive CD4(+) T cells in resistant mice treated once with anti-IL-12 or -IFN-gamma antibodies at the onset of infection with Leishmania major instructs Th2 cell development, resulting in nonhealing lesions

Rapid production of IL-4 by Leishmania homolog of mammalian RACK1 (LACK)-reactive CD4(+) T cells expressing the V beta 4-V alpha 8 TCR chains has been shown to drive aberrant Th2 cell development and susceptibility to Leishmania major in BALB/c mice. In contrast, mice from resistant strains fail to express this early IL-4 response. However, administration of either anti-IL-12 or -IFN-gamma at the initiation of infection allows the expression of this early IL-4 response in resistant mice. In this work we show that Leishmania homolog of mammalian RACK1-reactive CD4(+) T cells also expressing the V beta 4-V alpha 8 TCR chains are the source of the early IL-4 response to L. major in resistant mice given anti-IL-12 or -IFN-gamma Abs only at the onset of infection. Strikingly, these cells were found to be required for the reversal of the natural resistance of C57BL/6 mice following a single administration of anti-IL-12 or -IFN-gamma Abs. Together these results suggest that a deficiency in mechanisms capable of down-regulating the early IL-4 response to L. major contributes to the exquisite susceptibility of BALB/c mice to L. major.     

Sequential priming with simian immunodeficiency virus (SIV) DNA vaccines, with or without encoded cytokines, and a replicating adenovirus-SIV recombinant followed by protein boosting does not control a pathogenic SIVmac251 mucosal challenge

Previously, combination DNA/nonreplicating adenovirus (Ad)- or poxvirus-vectored vaccines have strongly protected against SHIV(89.6P), DNAs expressing cytokines have modulated immunity elicited by DNA vaccines, and replication-competent Ad-recombinant priming and protein boosting has strongly protected against simian immunodeficiency virus (SIV) challenge. Here we evaluated a vaccine strategy composed of these promising components. Seven rhesus macaques per group were primed twice with multigenic SIV plasmid DNA with or without interleukin-12 (IL-12) DNA or IL-15 DNA. After a multigenic replicating Ad-SIV immunization, all groups received two booster immunizations with SIV gp140 and SIV Nef protein. Four control macaques received control DNA plasmids, empty Ad vector, and adjuvant. All vaccine components were immunogenic, but the cytokine DNAs had little effect. Macaques that received IL-15-DNA exhibited higher peak anti-Nef titers, a more rapid anti-Nef anamnestic response postchallenge, and expanded CD8(CM) T cells 2 weeks postchallenge compared to the DNA-only group. Other immune responses were indistinguishable between groups. Overall, no protection against intrarectal challenge with SIV(mac251) was observed, although immunized non-Mamu-A*01 macaques as a group exhibited a statistically significant 1-log decline in acute viremia compared to non-Mamu-A*01 controls. Possible factors contributing to the poor outcome include administration of cytokine DNAs to sites different from the Ad recombinants (intramuscular and intratracheal, respectively), too few DNA priming immunizations, a suboptimal DNA delivery method, failure to ensure delivery of SIV and cytokine plasmids to the same cell, and instability and short half-life of the IL-15 component. Future experiments should address these issues to determine if this combination approach is able to control a virulent SIV challenge.     

Induction of genital immunity by DNA priming and intranasal booster immunization with a replication-defective adenoviral recombinant.

Mice immunized through different routes such as i.m., intradermally, or intratracheally with a DNA vaccine to rabies virus developed high titers of serum Ab but only borderline levels of mucosal Abs determined from vaginal secretions. DNA vaccines given by either route enhanced vaginal IgA and IgG2a secretion upon a subsequent intranasal booster immunization with an E1-deleted adenoviral recombinant expressing the same Ag of rabies virus. DNA vaccine priming reduced the Ab response to the adenoviral Ags and counterbalanced the impaired B cell response to the rabies virus Ag expressed by the adenoviral recombinant in mice preimmune to adenovirus. The vaginal B cell response could further be enhanced by using the Th2-type cytokines IL-4 or IL-5 as genetic adjuvants concomitantly with the DNA vaccine before intranasal booster immunization with the recombinant vaccine.     

Intranasal HIV-1-gp160-DNA/gp41 peptide prime-boost immunization regimen in mice results in long-term HIV-1 neutralizing humoral mucosal and systemic immunity

An intranasal DNA vaccine prime followed by a gp41 peptide booster immunization was compared with gp41 peptide and control immunizations. Serum HIV-1-specific IgG and IgA as well as IgA in feces and vaginal and lung secretions were detected after immunizations. Long-term humoral immunity was studied for up to 12 mo after the booster immunization by testing the presence of HIV-1 gp41- and CCR5-specific Abs and IgG/IgA-secreting B lymphocytes in spleen and regional lymph nodes in immunized mice. A long-term IgA-specific response in the intestines, vagina, and lungs was obtained in addition to a systemic immune response. Mice immunized only with gp41 peptides and L3 adjuvant developed a long-term gp41-specific serum IgG response systemically, although over a shorter period (1-9 mo), and long-term mucosal gp41-specific IgA immunity. HIV-1-neutralizing serum Abs were induced that were still present 12 mo after booster immunization. HIV-1 SF2-neutralizing fecal and lung IgA was detectable only in the DNA-primed mouse groups. Intranasal DNA prime followed by one peptide/L3 adjuvant booster immunization, but not a peptide prime followed by a DNA booster, was able to induce B cell memory and HIV-1-neutralizing Abs for at least half of a mouse's life span.     

BCG expressing LCR1 of Leishmania chagasi induces protective immunity in susceptible mice

Cellular immune responses are required for protective immunity against Leishmania chagasi. Immunization strategies using live intracellular bacteria (e.g., bacille-Calmette Guerin strain of Mycobacterium bovis) expressing recombinant antigens can induce cellular immune responses to these antigens. Previous studies demonstrated that the L. chagasi antigen LCR1 stimulates IFN-gamma production from T cells of infected BALB/c mice, and immunization with recombinant LCR1 partially protects against L. chagasi infection. To determine whether live bacteria could enhance the immunization potential of LCR1, we engineered BCG expressing LCR1 (BCG-LCR1). Subcutaneous immunization with BCG-LCR1, but not with BCG containing plasmid only (BCG-pMV261), elicited better protective immunity against L. chagasi infection than LCR1 protein alone. BCG-LCR1 administered intraperitoneally did not protect. Splenocytes from mice immunized s.c. with either BCG-LCR1 or BCG-pMV261 and then infected with L. chagasi promastigotes had increased antigen-induced IFN-gamma and reduced IL-10 production compared to splenocytes of control mice. We propose that BCG-LCR1 promotes a Th1-type protective immune response, and it may be a useful component of a Leishmania vaccine.