Complete protection against P. berghei malaria upon heterologous prime/boost immunization against circumsporozoite protein employing Salmonella type III secretion system and Bordetella adenylate cyclase toxoid

Sterile immunity against malaria can be achieved by the induction of IFNgamma-producing CD8(+) T cells that target infected hepatocytes presenting epitopes of the circumsporozoite protein (CSP). In the present study we evaluate the protective efficacy of a heterologous prime/boost immunization protocol based on the delivery of the CD8(+) epitope of Plasmodium berghei CSP into the MHC class I presentation pathway, by either a type III secretion system of live recombinant Salmonella and/or by direct translocation of a recombinant Bordetella adenylate cyclase toxoid fusion (ACT-CSP) into the cytosol of professional antigen-presenting cells (APCs). A single intraperitoneal application of the recombinant ACT-CSP toxoid, as well as a single oral immunization with the Salmonella vaccine, induced a specific CD8(+) T cell response, which however conferred only a partial protection on mice against a subsequent sporozoite challenge. In contrast, a heterologous prime/boost vaccination with the live Salmonella followed by ACT-CSP led to a significant enhancement of the CSP-specific T cell response and induced complete protection in all vaccinated mice.

Head-to-Head Comparison of Poxvirus NYVAC and ALVAC Vectors Expressing Identical HIV-1 Clade C Immunogens in Prime-Boost Combination with Env Protein in Nonhuman Primates.

UNLABELLED: We compared the HIV-1-specific cellular and humoral immune responses elicited in rhesus macaques immunized with two poxvirus vectors (NYVAC and ALVAC) expressing the same HIV-1 antigens from clade C, Env gp140 as a trimeric cell-released protein and a Gag-Pol-Nef polyprotein as Gag-induced virus-like particles (VLPs) (referred to as NYVAC-C and ALVAC-C). The immunization protocol consisted of two doses of the corresponding poxvirus vector plus two doses of a combination of the poxvirus vector and a purified HIV-1 gp120 protein from clade C. This immunogenicity profile was also compared to that elicited by vaccine regimens consisting of two doses of the ALVAC vector expressing HIV-1 antigens from clades B/E (ALVAC-vCP1521) plus two doses of a combination of ALVAC-vCP1521 and HIV-1 gp120 protein from clades B/E (similar to the RV144 trial regimen) or clade C. The results showed that immunization of macaques with NYVAC-C stimulated at different times more potent HIV-1-specific CD4(+) T-cell responses and induced a trend toward higher-magnitude HIV-1-specific CD8(+) T-cell immune responses than did ALVAC-C. Furthermore, NYVAC-C induced a trend toward higher levels of binding IgG antibodies against clade C HIV-1 gp140, gp120, or murine leukemia virus (MuLV) gp70-scaffolded V1/V2 and toward best cross-clade-binding IgG responses against HIV-1 gp140 from clades A, B, and group M consensus, than did ALVAC-C. Of the linear binding IgG responses, most were directed against the V3 loop in all immunization groups. Additionally, NYVAC-C and ALVAC-C also induced similar levels of HIV-1-neutralizing antibodies and antibody-dependent cellular cytotoxicity (ADCC) responses. Interestingly, binding IgA antibody levels against HIV-1 gp120 or MuLV gp70-scaffolded V1/V2 were absent or very low in all immunization groups. Overall, these results provide a comprehensive survey of the immunogenicity of NYVAC versus ALVAC expressing HIV-1 antigens in nonhuman primates and indicate that NYVAC may represent an alternative candidate to ALVAC in the development of a future HIV-1 vaccine. IMPORTANCE: The finding of a safe and effective HIV/AIDS vaccine immunogen is one of the main research priorities. Here, we generated two poxvirus-based HIV vaccine candidates (NYVAC and ALVAC vectors) expressing the same clade C HIV-1 antigens in separate vectors, and we analyzed in nonhuman primates their immunogenicity profiles. The results showed that immunization with NYVAC-C induced a trend toward higher HIV-1-specific cellular and humoral immune responses than did ALVAC-C, indicating that this new NYVAC vector could be a novel optimized HIV/AIDS vaccine candidate for human clinical trials.

CD8(+) T-cell-mediated immunity against malaria: a novel heterologous prime-boost strategy

Evaluation of: Rodriguez D, Gonzalez-Aseguinolaza G, Rodriguez JR et al. Vaccine efficacy against malaria by the combination of porcine parvovirus-like particles and vaccinia virus vectors expressing CS of Plasmodium. PLoS ONE 7(4), e34445 (2012). Recently, a vaccine against malaria was successfully tested in a human Phase III trial. The efficacy of this vaccine formulation, based on the Plasmodium falciparum circumsporozoite protein, was approximately 50% and correlated with the presence of antibodies specific to the infective stages of the malaria parasites. Different strategies are being pursued to improve vaccine efficacy levels. One such strategy is the induction of specific cytotoxic T cells that can destroy the intracellular hepatocyte stages of the malaria parasite. In this study, a novel vaccination protocol was developed to elicit strong immune responses mediated by CD8(+) cytotoxic cells specific to the circumsporozoite protein. As proof-of-concept, the authors used the rodent malaria Plasmodium yoelii parasite. The vaccination strategy consisted of a heterologous prime-boost vaccination regimen involving porcine parvovirus-like particles for priming and the modified vaccinia virus Ankara for the booster immunization, both of which expressed the immunodominant CD8 epitope of the P. yoelii circumsporozoite protein. Results from this experimental model were extremely meaningful. This vaccination strategy led to a significant T-cell immune response mediated by CD8(+) multifunctional T effector and effector-memory cells. However, most importantly for the malaria vaccine development was the fact that following a sporozoite challenge, immunized mice eliminated more than 97% of the malaria parasites during the hepatocyte stages. These results confirm and extend a vast body of knowledge showing that a heterologous prime-boost vaccination strategy can elicit strong CD8(+) T-cell-mediated protective immunity and may increase the efficacy of malaria vaccines.

Prime-boost vaccination with plasmid and adenovirus gene vaccines control HER2/neu+ metastatic breast cancer in mice.

INTRODUCTION: Once metastasis has occurred, the possibility of completely curing breast cancer is unlikely, particularly for the 30 to 40% of cancers overexpressing the gene for HER2/neu. A vaccine targeting p185, the protein product of the HER2/neu gene, could have therapeutic application by controlling the growth and metastasis of highly aggressive HER2/neu+ cells. The purpose of this study was to determine the effectiveness of two gene vaccines targeting HER2/neu in preventive and therapeutic tumor models. METHODS: The mouse breast cancer cell line A2L2, which expresses the gene for rat HER2/neu and hence p185, was injected into the mammary fat pad of mice as a model of solid tumor growth or was injected intravenously as a model of lung metastasis. SINCP-neu, a plasmid containing Sindbis virus genes and the gene for rat HER2/neu, and Adeno-neu, an E1,E2a-deleted adenovirus also containing the gene for rat HER2/neu, were tested as preventive and therapeutic vaccines. RESULTS: Vaccination with SINCP-neu or Adeno-neu before tumor challenge with A2L2 cells significantly inhibited the growth of the cells injected into the mammary fat or intravenously. Vaccination 2 days after tumor challenge with either vaccine was ineffective in both tumor models. However, therapeutic vaccination in a prime-boost protocol with SINCP-neu followed by Adeno-neu significantly prolonged the overall survival rate of mice injected intravenously with the tumor cells. Naive mice vaccinated using the same prime-boost protocol demonstrated a strong serum immunoglobulin G response and p185-specific cellular immunity, as shown by the results of ELISPOT (enzyme-linked immunospot) analysis for IFNgamma. CONCLUSION: We report herein that vaccination of mice with a plasmid gene vaccine and an adenovirus gene vaccine, each containing the gene for HER2/neu, prevented growth of a HER2/neu-expressing breast cancer cell line injected into the mammary fat pad or intravenously. Sequential administration of the vaccines in a prime-boost protocol was therapeutically effective when tumor cells were injected intravenously before the vaccination. The vaccines induced high levels of both cellular and humoral immunity as determined by in vitro assessment. These findings indicate that clinical evaluation of these vaccines, particularly when used sequentially in a prime-boost protocol, is justified.

Restoration of anti-tetanus toxoid responses in patients initiating highly active antiretroviral therapy with or without a boost immunization: an INITIO substudy.

INITIO is an open-labelled randomized trial evaluating first-line therapeutic strategies for human immunodeficiency virus-1 (HIV-1) infection. In an immunology substudy a tetanus toxoid booster (TTB) immunization was planned for 24 weeks after initiation of highly active antiretroviral therapy (HAART). All patients had received tetanus toxoid immunization in childhood. Generation of proliferative responses to tetanus toxoid was compared in two groups of patients, those receiving a protease inhibitor (PI)-sparing regimen (n = 21) and those receiving a PI-containing (n = 54) regimen. Fifty-two participants received a TTB immunization [PI-sparing (n = 15), PI-containing (n = 37)] and 23 participants did not [PI-sparing (n = 6) or PI-containing (n = 17)]. Cellular responses to tetanus antigen were monitored by lymphoproliferation at time of immunization and every 24 weeks to week 156. Proportions with a positive response (defined as stimulation index > or = 3 and Delta counts per minute > or = 3000) were compared at weeks 96 and 156. All analyses were intent-to-treat. Fifty-two participants had a TTB immunization at median 25 weeks; 23 patients did not. At weeks 96 and 156 there was no evidence of a difference in tetanus-specific responses, between those with or without TTB immunization (P = 0.2, P = 0.4). There was no difference in the proportion with response between those with PI-sparing or PI-containing regimens at both time-points (P = 0.8, P = 0.7). The proliferative response to tetanus toxoid was unaffected by initial HAART regimen. Anti-tetanus responses appear to reconstitute eventually in most patients over 156 weeks when treated successfully with HAART, irrespective of whether or not a TTB immunization has been administered.

Heterologous prime-boost regimen adenovector 35-circumsporozoite protein vaccine/recombinant Bacillus Calmette-Guérin expressing the Plasmodium falciparum circumsporozoite induces enhanced long-term memory immunity in BALB/c mice.

BACKGROUND: Sustained antibody levels are a hallmark of immunity against many pathogens, and induction of long-term durable antibody titers is an essential feature of effective vaccines. Heterologous prime-boost approaches with vectors are optimal strategies to improve a broad and prolonged immunogenicity of malaria vaccines. RESULTS: In this study, we demonstrate that the heterologous prime-boost regimen Ad35-CS/BCG-CS induces stronger immune responses by enhancing type 1 cellular producing-cells with high levels of CSp-specific IFN-γ and cytophilic IgG2a antibodies as compared to a homologous BCG-CS and a heterologous BCG-CS/CSp prime-boost regimen. Moreover, the heterologous prime-boost regimen elicits the highest level of LLPC-mediated immune responses. CONCLUSION: The increased IFN-γ-producing cell responses induced by the combination of Ad35-CS/BCG-CS and sustained type 1 antibody profile together with high levels of LLPCs may be essential for the development of long-term protective immunity against liver-stage parasites.

Bacille Calmette-Guérin/DNAhsp65 prime-boost is protective against diabetes in non-obese diabetic mice but not in the streptozotocin model of type 1 diabetes

Type I diabetes is a disease caused by autoimmune destruction of the beta cells in the pancreas that leads to a deficiency in insulin production. The aim of this study was to evaluate the prophylactic potential of a prime-boost strategy involving bacille Calmette-Guérin (BCG) and the pVAXhsp65 vaccine (BCG/DNAhsp65) in diabetes induced by streptozotocin (STZ) in C57BL/6 mice and also in spontaneous type 1 diabetes in non-obese diabetic (NOD) mice. BCG/DNAhsp65 vaccination in NOD mice determined weight gain, protection against hyperglycaemia, decreased islet inflammation, higher levels of cytokine production by the spleen and a reduced number of regulatory T cells in the spleen compared with non-immunized NOD mice. In the STZ model, however, there was no significant difference in the clinical parameters. Although this vaccination strategy did not protect mice in the STZ model, it was very effective in NOD mice. This is the first report demonstrating that a prime-boost strategy could be explored as an immunomodulatory procedure in autoimmune diseases. © 2013 British Society for Immunology.

Immunization with a circumsporozoite epitope fused to Bordetella pertussis adenylate cyclase in conjunction with cytotoxic T-lymphocyte-associated antigen 4 blockade confers protection against Plasmodium berghei liver-stage malaria

The adenylate cyclase toxoid (ACT) of Bordetella pertussis is capable of delivering its N-terminal catalytic domain into the cytosol of CD11b-expressing professional antigen-presenting cells such as myeloid dendritic cells. This allows delivery of CD8+ T-cell epitopes to the major histocompatibility complex (MHC) class I presentation pathway. Recombinant detoxified ACT containing an epitope of the Plasmodium berghei circumsporozoite protein (CSP), indeed, induced a specific CD8+ T-cell response in immunized mice after a single application, as detected by MHC multimer staining and gamma interferon (IFN-gamma) ELISPOT assay. This CSP-specific response could be significantly enhanced by prime-boost immunization with recombinant ACT in combination with anti-CTLA-4 during the boost immunization. This increased response was accompanied by complete protection in a number of mice after a challenge with P. berghei sporozoites. Transient blockade of CTLA-4 may overcome negative regulation and hence provide a strategy to enhance the efficacy of a vaccine by amplifying the number of responding T cells.

Leukotrienes are not essential for the efficacy of a heterologous vaccine against Mycobacterium tuberculosis infection

Leukotrienes are reported to be potent proinflammatory mediators that play a role in the development of several inflammatory diseases such as asthma, rheumatoid arthritis and periodontal disease. Leukotrienes have also been associated with protection against infectious diseases. However, the role of leukotrienes in Mycobacterium tuberculosis infection is not understood. To answer this question, we studied the role of leukotrienes in the protective immune response conferred by prime-boost heterologous immunization against tuberculosis. We immunized BALB/c mice (4-11/group) with subcutaneous BCG vaccine (1 x 10(5) M. bovis BCG) (prime) followed by intramuscular DNA-HSP65 vaccine (100 µg) (boost). During the 30 days following the challenge, the animals were treated by gavage daily with MK-886 (5 mg·kg-1·day-1) to inhibit leukotriene synthesis. We showed that MK-886-treated mice were more susceptible to M. tuberculosis infection by counting the number of M. tuberculosis colony-forming units in lungs. The histopathological analysis showed an impaired influx of leukocytes to the lungs of MK-886-treated mice after infection, confirming the involvement of leukotrienes in the protective immune response against experimental tuberculosis. However, prime-boost-immunized mice treated with MK-886 remained protected after challenge with M. tuberculosis, suggesting that leukotrienes are not required for the protective effect elicited by immunization. Protection against M. tuberculosis challenge achieved by prime-boost immunization in the absence of leukotrienes was accompanied by an increase in IL-17 production in the lungs of these animals, as measured by ELISA. Therefore, these data suggest that the production of IL-17 in MK-886-treated, immunized mice could contribute to the generation of a protective immune response after infection with M. tuberculosis.

Alzheimer's disease: is a vaccine possible?

The cause of Alzheimer's disease is still unknown, but the disease is distinctively characterized by the accumulation of β-amyloid plaques and neurofibrillary tangles in the brain. These features have become the primary focus of much of the research looking for new treatments for the disease, including immunotherapy and vaccines targeting β-amyloid in the brain. Adverse effects observed in a clinical trial based on the β-amyloid protein were attributed to the presence of the target antigen and emphasized the relevance of finding safer antigen candidates for active immunization. For this kind of approach, different vaccine formulations using DNA, peptide, and heterologous prime-boost immunization regimens have been proposed. Promising results are expected from different vaccine candidates encompassing B-cell epitopes of the β-amyloid protein. In addition, recent results indicate that targeting another protein involved in the etiology of the disease has opened new perspectives for the effective prevention of the illness. Collectively, the evidence indicates that the idea of finding an effective vaccine for the control of Alzheimer's disease, although not without challenges, is a possibility.

Boosting systemic and secreted antibody responses in mice orally immunized with recombinant Bacillus subtilis strains following parenteral priming with a DNA vaccine encoding the enterotoxigenic Escherichia coli (ETEC) CFA/I fimbriae B subunit

Recombinant Bacillus subtilis strains, either spores or vegetative cells, may be employed as safe and low cost orally delivered live vaccine vehicles. In this study, we report the use of an orally delivered B. subtilis vaccine strain to boost systemic and secreted antibody responses in mice i.m. primed with a DNA vaccine encoding the structural subunit (CfaB) of the CFA/I fimbriae encoded by enterotoxigenic Escherichia coli (ETEC), an important etiological agent of diarrhea among travelers and children living in endemic regions. DBA/2 female mice submitted to the prime-boost immunization regimen developed synergic serum (IgG) and mucosal (IgA) antibody responses to the target CfaB antigen. Moreover, in contrast to mice immunized only with one vaccine formulation, sera harvested from prime-boosted vaccinated individuals inhibited adhesion of ETEC cells to human red blood cells. Additionally, vaccinated dams conferred full passive protection to suckling newborn mice challenged with a virulent ETEC strain. Taken together the present results further demonstrate the potential use of recombinant B. subtilis strains as an alternative live vaccine vehicle. (C) 2008 Elsevier Ltd. All rights reserved.

Leukotrienes are not essential for the efficacy of a heterologous vaccine against Mycobacterium tuberculosis infection

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Use of Recombinant Virus Replicon Particles for Vaccination against Mycobacterium ulcerans Disease.

Buruli ulcer, caused by infection with Mycobacterium ulcerans, is a necrotizing disease of the skin and subcutaneous tissue, which is most prevalent in rural regions of West African countries. The majority of clinical presentations seen in patients are ulcers on limbs that can be treated by eight weeks of antibiotic therapy. Nevertheless, scarring and permanent disabilities occur frequently and Buruli ulcer still causes high morbidity. A vaccine against the disease is so far not available but would be of great benefit if used for prophylaxis as well as therapy. In the present study, vesicular stomatitis virus-based RNA replicon particles encoding the M. ulcerans proteins MUL2232 and MUL3720 were generated and the expression of the recombinant antigens characterized in vitro. Immunisation of mice with the recombinant replicon particles elicited antibodies that reacted with the endogenous antigens of M. ulcerans cells. A prime-boost immunization regimen with MUL2232-recombinant replicon particles and recombinant MUL2232 protein induced a strong immune response but only slightly reduced bacterial multiplication in a mouse model of M. ulcerans infection. We conclude that a monovalent vaccine based on the MUL2232 antigen will probably not sufficiently control M. ulcerans infection in humans.

Augmentation de l'immunogénicité de la nucléoprotéine de l'influenza par multimérisation in vitro

Les travaux effectués au cours de ce mémoire ont permis de développer une alternative aux vaccins présentement utilisés contre le virus de l’influenza. Nous avons utilisé la nucléoprotéine (NP) de l’influenza comme base vaccinale puisque cette protéine est conservée chez les souches d’influenza A et qu’elle possède un potentiel de protection croisée. Nous avons montré que la multimérisation de la NP grâce à un gabarit d’ARN permet d’augmenter son immunogenicité. Cette multimérisation en pseudo-nucléoparticule virale (NLP) a augmenté la réponse humorale et cellulaire spécifique à NP et l’ajout d’un adjuvant (PAL) a permis d’amplifier davantage la réponse humorale contre NP. Une dose du vaccin candidat NLP-PAL n’a pas réussi à protéger des souris contre une infection létale avec une souche homotypique d’influenza. Cependant, des résultats avec un régime de deux immunisations montrent des résultats encourageants qui permettent d’espérer une protection envers une infection virale.

Prime and boost vaccination against HER2/neu in breast cancer

Breast cancer is the most common cancer among women with approximately 180,000 new cases being diagnosed yearly in the United States (1). HER2/neu gene amplification and subsequent protein overexpression is found in 20–30% of breast cancer patients and can lead to the promotion of various metastasis-related properties (2–4) and/or resistance to cancer therapies such as chemotherapy and radiation (5). ^ The protein product of the HER2/neu gene, p185, is a proven target for immunological therapy. Recently, passive immunotherapy with the monoclonal antibody Trastuzumab® has validated an immunological approach to HER2/neu+ breast cancer. Immunity to HER2/ neu, when found in breast cancer patients, is of low magnitude. Vaccination-induced HER2/neu-specific antibodies and HER2/neu-specific cytotoxic T cells could result in long-lived immunity with therapeutic benefit. Many features of DNA vaccines and attenuated viral vectors may contribute to the efficacy of prime-boost vaccination. In particular, vaccines capable of eliciting strong cell-mediated immunity are thought to hold the greatest promise for control of cancer (6–9). ^ To optimize cellular immunization to HER2/neu in my study, the HER2/neu gene was presented to the immune system using a priming vector followed by a second vector used as the boost. In both animals and humans, priming with DNA and boosting with a poxviruses, vaccinia or canarypox appears to be particularly promising for induction of a broad immune responses (10). ^ I tested three gene vaccines encoding the HER2/neu gene: (1) a plasmid, SINCP, that contains part of the genome of Sindbis virus; (2) Viral Replicon Particles (VRP) of Venezuela Equine Encephalitis virus (VEE) and (3) E1/E2a-deleted human Type 5 Adenovirus. In SINCP and the VRP, the caspid and envelope genes of the virus were deleted and replaced with the gene for HER2/neu. SINCP-neu, VRP- neu and Adeno-neu when used alone were effective vaccines protecting healthy mice from challenge with a breast cancer cell line injected in the mammary fat pad or injected i.v. to induce experimental lung metastasis. However, SINCP-neu, VRP-neu or Adeno-neu when used alone were not able to prolong survival of mice in therapeutic models in which vaccination occurred after injection of a breast cancer cell line. ^ When the vaccines were combined in a mixed regimen of a SINCP- neu prime VRP-neu or Adeno-neu boost, there was a significant difference in tumor growth and survival in the therapeutic vaccine models. In vitro assays demonstrated that vaccination with each of the three vaccines induced IgG specific for p185, the gene product of HER2/neu, induced p185-specific T lymphocytes, as measured by tetramer analysis. Vaccination also induced intracellular INF-γ and a positive ELISPOT assay. These findings indicate that SINCP-neu, VRP-neu and Adeno-neu, used alone or in combination, may have clinical potential as adjuvant immunotherapy for the treatment of HER2/neu-expressing tumors. ^