A prime-boost immunisation regimen using DNA followed by recombinant modified vaccinia virus Ankara induces strong cellular immune responses against the Plasmodium falciparum TRAP antigen in chimpanzees

Two chimpanzees were vaccinated intramuscularly against malaria using plasmid DNA expressing the pre-erythrocytic antigens thrombospondin related adhesion protein (PfTRAP) and liver stage specific antigen-1 (PfLSA-1) of Plasmodium falciparum together with GM-CSF protein. A recombinant modified vaccinia virus Ankara (MVA) expressing PfTRAP was injected intramuscularly 6 weeks later to boost the immune response. This sequence of antigen delivery induced a specific and long-lasting T cell and antibody response to PfTRAP as detected by ELISPOT assay and ELISA. Antibody responses were detected after four DNA injections, and were boosted by injection of recombinant MVA expressing PfTRAP. Interferon-gamma secreting antigen-specific T cells were detected in both animals, but only after boosting with recombinant MVA. By screening a panel of PfTRAP-derived peptides, an epitope was identified that was recognized by cytotoxic T lymphocytes in one of the chimpanzees studied. T cells specific for this epitope were present in PBMCs and liver-infiltrating lymphocytes at a frequency of between 1 in 200 and 1 in 500. The high immunogenicity of this prime-boost regimen in chimpanzees supports further assessment of this delivery strategy for the induction of protection against P. falciparum malaria in humans.     

Gene gun intradermal DNA immunization followed by boosting with modified vaccinia virus Ankara: enhanced CD8+ T cell immunogenicity and protective efficacy in the influenza and malaria models

In influenza and malaria, CD8+ T cells play an important role in protective immunity in mice. An immunization strategy consisting of DNA priming followed by boosting with recombinant modified vaccinia virus Ankara (MVA) induces complete protection, associated with high levels of CD8+ T cells, against Plasmodium berghei sporozoite challenge in mice. Intradermal delivery of DNA with a gene gun requires smaller amounts of DNA than intramuscular injection, in order to induce similar levels of immune responses. The present study compares both routes for the induction of specific CD8+ T cell responses and protection using different prime–boost immunization regimes in the influenza and the malaria models. In the DNA/MVA regime, equally high CD8+ T cell responses and levels of protection are achieved using ten times less DNA when delivered with a gene gun compared to intramuscular injection.     

Enhanced CD8 T cell immunogenicity and protective efficacy in a mouse malaria model using a recombinant adenoviral vaccine in heterologous prime-boost immunisation regimes.

Recombinant replication-defective adenovirus expressing the CS gene from Plasmodium berghei (Ad-PbCS) was found to induce a strong CD8(+) T cell response after intra-dermal or -muscular immunisation. Boosting of an adenovirus-primed immune response with the replication-impaired poxvirus, modified vaccinia virus Ankara (MVA) led to enhanced immunogenicity and substantial protective efficacy. The recombinant adenoviral vaccine was capable of boosting to protective levels a CD8(+) T cell response primed by either a plasmid DNA vaccine, a recombinant Ty virus-like particle vaccine or recombinant MVA each expressing the same epitope or antigen. Complete protective efficacy after intradermal immunisation was observed with the adenovirus prime-MVA boost regime. This study identifies recombinant replication-defective adenovirus as an alternative to recombinant replication-defective poxviruses as boosting agents for the induction of strong protective CD8(+) T cell responses.     

Safety of recombinant fowlpox strain FP9 and modified vaccinia virus Ankara vaccines against liver-stage P. falciparum malaria in non-immune volunteers

The ability to generate potent antigen-specific T cell responses by vaccination has been a major hurdle in vaccinology. Vaccinia virus and avipox viruses have been shown to be capable of expressing antigens in mammalian cells and can induce a protective immune response against several mammalian pathogens. We report on two such vaccine constructs, modified vaccinia virus Ankara and FP9 (an attenuated fowlpox virus) both expressing the pre-erythrocytic malaria antigen thrombospondin-related adhesion protein and a string of CD8+ epitopes (ME-TRAP). In prime-boost combinations in a mouse model MVA and FP9 are highly immunogenic and induce substantial protective efficacy. A series of human clinical trials using the recombinant MVA and FP9 malaria vaccines encoding ME-TRAP, both independently and in prime-boost combinations with or without the DNA vaccine DNA ME-TRAP, has shown them to be both immunogenic for CD8+ T cells and capable of inducing protective efficacy. We report here a detailed analysis of the safety profiles of these viral vectors and show that anti-vector antibody responses induced by the vectors are generally low to moderate. We conclude that these vectors are safe and show acceptable side effect profiles for prophylactic vaccination.     

Human CD8+ T cells mediate protective immunity induced by a human malaria vaccine in human immune system mice

A number of studies have shown that CD8+ T cells mediate protective anti-malaria immunity in a mouse model. However, whether human CD8+ T cells play a role in protection against malaria remains unknown. We recently established human immune system (HIS) mice harboring functional human CD8+ T cells (HIS-CD8 mice) by transduction with HLA-A10201 and certain human cytokines using recombinant adeno-associated virus-based gene transfer technologies. These HIS-CD8 mice mount a potent, antigen-specific HLA-A10201-restricted human CD8+ T-cell response upon immunization with a recombinant adenovirus expressing a human malaria antigen, the Plasmodium falciparum circumsporozoite protein (PfCSP), termed AdPfCSP. In the present study, we challenged AdPfCSP-immunized HIS-CD8 mice with transgenic Plasmodium berghei sporozoites expressing full-length PfCSP and found that AdPfCSP-immunized (but not naïve) mice were protected against subsequent malaria challenge. The level of the HLA-A10201-restricted, PfCSP-specific human CD8+ T-cell response was closely correlated with the level of malaria protection. Furthermore, depletion of human CD8+ T cells from AdPfCSP-immunized HIS-CD8 mice almost completely abolished the anti-malaria immune response. Taken together, our data show that human CD8+ T cells mediate protective anti-malaria immunity in vivo.     

Immunogenicity of Ty-VLP bearing a CD8(+) T cell epitope of the CS protein of P. yoelii: enhanced memory response by boosting with recombinant vaccinia virus

We characterized the immunogenicity of the hybrid Ty-virus-like carrying the CD8(+) T cell epitope (SYVPSAEQI) of the circumsporozoite (CS) protein of Plasmodium yoelii (TyCS-VLP), a rodent malaria parasite. Balb/c mice were immunized with hybrid TyCS-VLP, and their CS-specific CD8(+) T cell response was quantitatively evaluated with the ELISPOT assay, based on the enumeration of epitope specific gamma-interferon secreting CD8(+) T cell. A single immunization with the TyCS-VLP by a variety of routes and doses indicated that the maximal response occurred in mice, which were immunized with 50 micrograms of these particles, administered via intramuscular. Combined immunization of mice with this TyCS-VLP followed by recombinant vaccinia virus expressing the entire P. yoelii CS protein (VacPyCS) or irradiated sporozoites, induced high levels of IFN-gamma-producing cells. The immunization regime, priming with TyCS-VLP and boosting with VacPyCS generated a potent protective immune response, which strongly inhibited P. yoelii liver stages development and protected 62% of the mice against a subsequent live P. yoelii sporozoite challenge.     

Recombinant modified vaccinia virus Ankara efficiently restimulates human cytotoxic T lymphocytes in vitro

The immunogenicity of recombinant modified vaccinia Ankara, a highly attenuated vaccinia virus, expressing influenza nucleoprotein (MVA-NP) and HIV-1 gag (MVA-gag) was investigated. Restimulation of peripheral blood lymphocytes of healthy subjects with MVA-NP led to expansion of CTL with specificity for known NP epitopes. These CTL efficiently lysed NP peptide-pulsed targets and released interferon-gamma (IFN-gamma) on contact with epitope peptide. MVA-NP-stimulated CTL specific for the HLA-B8 epitope, NP380-88, stained with a tetrameric complex of HLA-B8 refolded with the NP380-88 peptide and anti-CD8 antibody on flow cytometry. CTL were also elicited from two HIV-1 seropositive donors by restimulation with MVA-HIV-1 gag and showed specificity for immunodominant gag epitopes. These data indicate that restimulation of human CTL with recombinant MVA is effective and suggest that MVA will elicit CTL responses in humans in vivo.     

Safety of DNA and modified vaccinia virus Ankara vaccines against liver-stage P. falciparum malaria in non-immune volunteers

A series of phase I clinical studies were conducted to evaluate the safety of plasmid DNA and modified vaccinia virus Ankara malaria vaccines. The vaccines each encoded a polyepitope string fused to whole Plasmodium falciparum TRAP antigen. Forty-three healthy adult volunteers received the vaccines alone or in DNA/MVA prime-boost combinations. The DNA vaccine was administered either intramuscularly by needle or intradermally by a needleless delivery device. The MVA vaccine was administered intradermally by needle. The vaccines were well-tolerated by all three routes and in various DNA/MVA immunisation regimes. There were no severe or serious adverse events.     

Immunogenicity of the candidate malaria vaccines FP9 and modified vaccinia virus Ankara encoding the pre-erythrocytic antigen ME-TRAP in 1-6 year old children in a malaria endemic area

In a phase 1 trial, 22 children in a malaria endemic area were immunised with candidate malaria vaccination regimes. The regimes used two recombinant viral vectors, attenuated fowlpox strain FP9 and modified vaccinia virus Ankara (MVA). Both encoded the pre-erythrocytic malaria antigen construct ME-TRAP. Strong T cell responses were detected by both ex vivo and cultured ELISpot assays. Data from phase 1 trials in adults on anti-vector responses raised by FP9 is presented. These responses partially cross-reacted with MVA, and detectably reduced the immunogenicity of vaccination with MVA. This prompted the comparison of half dose and full dose FP9 priming vaccinations in children. Regimes using half dose FP9 priming tended to be more immunogenic than full dose. The potential for enhanced immunogenicity with half doses of priming vectors warrants further investigation, and larger studies to determine protection against malaria in children are required.     

Comparing the safety and immunogenicity of a candidate TB vaccine MVA85A administered by intramuscular and intradermal delivery

Background

New vaccines to prevent tuberculosis are urgently needed. MVA85A is a novel viral vector TB vaccine candidate designed to boost BCG-induced immunity when delivered intradermally. To date, intramuscular delivery has not been evaluated. Skin and muscle have distinct anatomical and immunological properties which could impact upon vaccine-mediated cellular immunity.

Methods

We conducted a randomised phase I trial comparing the safety and immunogenicity of 1 × 10⁸ pfu MVA85A delivered intramuscularly or intradermally to 24 healthy BCG-vaccinated adults.

Results

Intramuscular and intradermal MVA85A were well tolerated. Intradermally-vaccinated subjects experienced significantly more local adverse events than intramuscularly-vaccinated subjects, with no difference in systemic adverse events. Both routes generated strong and sustained Ag85A-specific IFNγ T cell responses and induced multifunctional CD4+ T cells. The frequencies of CD4+ T cells expressing chemokine receptors CCR4, CCR6, CCR7 and CXCR3 induced by vaccination was similar between routes.

Conclusions

In this phase I trial the intramuscular delivery of MVA85A was well tolerated and induced strong, durable cellular immune responses in healthy BCG vaccinated adults, comparable to intradermal delivery. These findings are important for TB vaccine development and are of relevance to HIV, malaria, influenza and other intracellular pathogens for which T cell-inducing MVA-based vaccine platforms are being evaluated.     

Boosting BCG vaccination with MVA85A down-regulates the immunoregulatory cytokine TGF-beta1

In clinical trials recombinant-modified vaccinia virus Ankara expressing the Mycobacterium tuberculosis antigen 85A (MVA85A) induces approximately 10 times more effector T cells than any other recombinant MVA vaccine. We have found that in BCG primed subjects MVA85A vaccination reduces transforming growth factor beta 1 (TGF-beta1) mRNA in peripheral blood lymphocytes and reduces TGF-beta1 protein in the serum, but increases IFN-gamma ELISPOT responses to the recall antigen SK/SD. TGF-beta1 is essential for the generation of regulatory T cells and we see a correlation across vaccinees between CD4+CD25hiFoxP3+ cells and TGF-beta1 serum levels. This apparent ability to counteract regulatory T cell effects suggests a potential use of MVA85A as an adjuvant for less immunogenic vaccines.     

The action of falciparum malaria on the human and chimpanzee genomes compared: Absence of evidence for a genomic signature of malaria at HBB and G6PD in three subspecies of chimpanzee

The historical association between Plasmodium and primates has meant that many Plasmodium species have coevolved with specific primate hosts. However, unlike humans that are infected by species such as P. falciparum that cause severe malaria, many non-human primates are infected by Plasmodium species that only cause mild disease. Here we investigate whether the genomic signatures of plasmodial infection found in humans are also present in chimpanzees. We find no evidence of the major deleterious mutations at HBB (β-globin) and G6PD in chimpanzees that confer resistance to malaria caused by P. falciparum nor evidence of long-term balancing selection at these loci. Our knowledge of malaria prevalence and pathogenesis in wild chimpanzees is severely limited, but it may be the case that β-globin and G6PD variation are not adaptive in chimpanzees because malaria is rare and/or less detrimental in this species. Alternatively, chimpanzees may utilise mechanisms that are different from those of humans to protect against malaria.     

Enhanced CD8+ T cell response to HIV-1 env by combined immunization with influenza and vaccinia virus recombinants

With the aim to determine if immunization with two different live recombinant viral vectors could lead to an enhancement of the cellular immune response to HIV-1 antigens, we have characterized the CD8+ T cell response elicited against the V3 loop epitope from HIV-1 env protein in Balb/c mice immunized with either: a recombinant influenza virus (Flu-Env) expressing the V3 loop epitope from HIV-1 strain IIIB, a vaccinia virus recombinant (VV-Env) expressing the complete HIV-1-IIIB env protein, or a combination of both. The CD8+ T cell response, measured by the ELISPOT assay, in animals primed with Flu-Env and boosted with VV-Env was 5 to 6 times higher than in animals inoculated with either Flu-Env or VV-Env alone. Similar results were obtained with recombinant viruses expressing the V3 loop epitope or the complete env protein, respectively, from the MN strain of HIV-1. Our results indicate that the use of two different live vectors for priming and boosting has a synergistic effect on the immune response against HIV-1, and could represent a novel vaccination strategy against AIDS.     

Synergistic DNA-MVA prime-boost vaccination regimes for malaria and tuberculosis

T-cell-mediated responses against the liver-stage of Plasmodium falciparum are critical for protection in the human irradiated sporozoite model and several animal models. Heterologous prime-boost approaches, employing plasmid DNA and viral vector delivery of malarial DNA sequences, have proved particularly promising for maximising T-cell-mediated protection in animal models. The T-cell responses induced by this prime-boost regime, in animals and humans, are substantially greater than the sum of the responses induced by DNA or MVA vaccines used alone, leading to the term introduced here of "synergistic" prime-boost immunisation. The insert in our first generation clinical constructs is known as multiple epitope-thrombospondin-related adhesion protein (ME-TRAP). We have performed an extensive series of phase I/II trials evaluating various prime-boost combination regimens for delivery of ME-TRAP in over 500 malaria-na?ve and malaria-exposed individuals. The three delivery vectors are DNA, modified vaccinia virus Ankara (MVA) and, more recently, fowlpox strain 9 (FP9). Administration was intra-epidermal and intramuscular for DNA and intradermal for MVA and FP9. Doses of DNA ranged from 4 microg to 2mg. Doses of MVA were up to 1.5 x 10(8) plaque forming units (pfu) and of FP9, up to 1.0 x 10(8)pfu. Further trials employing bacille Calmette-Guérin (BCG) as the priming agent and MVA expressing antigen 85A of Mycobacterium tuberculosis as the boosting agent has extended the scope of synergistic prime-boost vaccination. In this review we summarise the safety, immunogenicity and efficacy results from these malaria and tuberculosis vaccine clinical trials.     

A phase 1, randomized,controlled dose-escalation study of EP-1300 polyepitope DNA vaccine against Plasmodium falciparum malaria administered via electroporation

Plasmodium falciparum malaria is one of the leading infectious causes of childhood mortality in Africa. EP-1300 is a polyepitope plasmid DNA vaccine expressing 38 cytotoxic T cell epitopes and 16 helper T cell epitopes derived from P. falciparum antigens expressed predominantly in the liver phase of the parasite’s life cycle. We performed a phase 1 randomized, placebo-controlled, dose escalation clinical trial of the EP-1300 DNA vaccine administered via electroporation using the TriGrid Delivery System device (Ichor Medical Systems). Although the delivery of the EP-1300 DNA vaccine via electroporation was safe, tolerability was less than that usually observed with standard needle and syringe intramuscular administration. This was primarily due to acute local discomfort at the administration site during electroporation. Despite the use of electroporation, the vaccine was poorly immunogenic. The reasons for the poor immunogenicity of this polyepitope DNA vaccine remain uncertain.Clinical Trials Registration: ClinicalTrials.gov NCT01169077.     

Protective immunity against heterologous challenge with encephalomyocarditis virus by VP1 DNA vaccination: effect of coinjection with a granulocyte-macrophage colony stimulating factor gene

For DNA vaccination studies, recombinant VP1 protein of encephalomyocarditis virus (EMCV) was produced from Escherichia coli, and eukaryotic VP1 expression vector, pCT-Gs-VP1, was generated and used as a DNA vaccine. Mice were immunized intramuscularly (i.m.) with pCT-Gs-VP1 in the presence or absence of plasmid DNA expressing granulocyte-macrophage colony stimulating factor (GM-CSF), and were subsequently analyzed for their anti-VP1 immune responses with recombinant VP1 in ELISA. Immunization of mice with pCT-Gs-VP1 resulted in VP1-specific immune response and 43% protection from subsequent lethal heterologous challenge of EMCV. Coinjection of mice with pCT-Gs-VP1 and plasmid DNA encoding GM-CSF was shown to increase the seroconversion rate of the immunized mice with a single DNA injection, and enhanced to a higher degree VP1-specific immunity, which appeared to result in better protection (about 80%) from lethal virus challenge. Thus, our results provide evidence for the potential use of GM-CSF to induce better immune response and resistance against viral infection in DNA vaccination.     

Mucosal and systemic immune responses induced after oral delivery of vaccinia virus recombinants

The immune responses elicited after oral delivery of vaccinia virus (VV) recombinants are not well defined. In this study we show with mice, that after oral administration of a VV recombinant expressing the luciferase reporter gene, VV gene expression takes place for several days in gut-associated lymphoid (GALT) tissues as well as in the spleen. After 14 days, a significant mucosal IgA response against VV was detected in vaginal and intestinal washings, as well as a systemic specific IgG response, which was principally of the IgG2a subclass. Furthermore, orally immunized mice developed cellular immune responses to VV (CD8+ T cells and T helper activities) in mesenteric lymph nodes (MLN) and spleen. Oral immunization with a VV recombinant expressing, either the envelope protein of HIV or beta-galactosidase, induced a specific immune response, locally and systemically, against gp120 and beta-gal. The cytokine pattern found in supernatants of spleen and MLN cells after stimulation with VV antigens or gp120 was clearly of type 1 cytokines. These studies demonstrate that VV recombinants administered by the oral route generate mucosal and systemic immune responses against antigens of the virus vector and to the recombinant products. These observations are of significance in the use of poxvirus vectors as vaccines.     

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.     

Intranasal immunization with synthetic recombinant vaccine containing multiple epitopes of influenza virus

The oligonucleotides coding for three epitopes (HA91-108, NP55-69, and NP 147-158) of influenza virus, stimulating B-cells, T-helper cells and cytotoxic T lymphocytes (CTLs), respectively, were previously employed for expressing each epitope in flagella that induced specific humoral and cellular immune responses. We have constructed new plasmids expressing all three epitopes as a single recombinant product. Two versions have been prepared-a longer one (Fla-HNN) comprising hybrid flagella containing the epitopes, and a shorter version (HNN). Immunization of BALB/c mice with either constructs induced significant humoral immune response against influenza virus. The mice immunized with these peptides also induced higher T-helper activity, including Th1 type-cytokine (IL-2 and IFN-gamma) release. In addition, the mice immunized with HNN peptide demonstrated significant protection against sublethal viral challenge. Furthermore, this vaccine fully protected mice from lethal challenge and enhanced their recovery process. Our results indicate that a single construct expressing multiple epitopes, which stimulate different arms of the immune system, might be an appropriate candidate when the synthetic recombinant vaccine approach is considered.     

Induction of protective antiviral cytotoxic T cells by a tubular structure capable of carrying large foreign sequences

Bluetongue virus (BTV) produces large numbers of tubules during infection which are formed by a single virus coded non-structural protein, NS1. The NS1 protein has been fused with full length green fluorescent protein (GFP) and was shown to retain the capacity to form tubules when expressed in heterologous expression systems. Moreover, recombinant purified chimeric tubules were demonstrated to be internalized by macrophages and dendritic cells. The ability of such chimeric tubules to induce protective cytotoxic T lymphocytes (CTL) responses has been assessed by generating chimeric tubules carrying a single CD8(+) T cell epitope from the lymphocytic choriomeningitis virus (LCMV) nucleoprotein. These chimeric tubules were recognized by MHC class I restricted T cell hybridoma in vitro and induced in vivo strong CD8(+) class I-restricted CTL responses in immunized mice. Further, the immunized mice were protected when challenged with a lethal dose of LCMV. This is the first study that demonstrates that the virus derived tubules synthesized by a recombinant non-structural protein carrying a single viral CTL epitope could induce protective immunity against a lethal viral challenge. Since recombinant tubules carrying large inserts can be purified at a large quantity from insect cells, they have potential to develop as safe multi-CTL vaccine delivery systems.