Characterization and biodistribution of under-employed gene therapy vector AAV7.

IMPORTANCE: The use of adeno-associated viruses (AAVs) as gene delivery vectors has vast potential for the treatment of many severe human diseases. Over one hundred naturally existing AAV capsid variants have been described and classified into phylogenetic clades based on their sequences. AAV8, AAV9, AAVrh.10, and other intensively studied capsids have been propelled into pre-clinical and clinical use, and more recently, marketed products; however, less-studied capsids may also have desirable properties (e.g., potency differences, tissue tropism, reduced immunogenicity, etc.) that have yet to be thoroughly described. These data will help build a broader structure-function knowledge base in the field, present capsid engineering opportunities, and enable the use of novel capsids with unique properties.

The presence of CpGs in AAV gene therapy vectors induces a plasmacytoid dendritic cell-like population very early after administration.

AAV-mediated gene transfer is a promising platform still plagued by potential host-derived, antagonistic immune responses to therapeutic components. CpG-mediated TLR9 stimulation activates innate immune cells and leads to cognate T cell activation and suppression of transgene expression. Here, we demonstrate that CpG depletion increased expression of an antibody transgene product by 2-3-fold as early as 24 h post-vector administration in mice. No significant differences were noted in anti-transgene product/ anti-AAV capsid antibody production or cytotoxic gene induction. Instead, CpG depletion significantly reduced the presence of a pDC-like myeloid cell population, which was able to directly bind the antibody transgene product via Fc-FcγR interactions. Thus, we extend the mechanisms of TLR9-mediated antagonism of transgene expression in AAV gene therapy to include the actions of a previously unreported pDC-like cell population.

Sex Difference Leads to Differential Gene Expression Patterns and Therapeutic Efficacy in Mucopolysaccharidosis IVA Murine Model Receiving AAV8 Gene Therapy.

Adeno-associated virus (AAV) vector-based therapies can effectively correct some disease pathology in murine models with mucopolysaccharidoses. However, immunogenicity can limit therapeutic effect as immune responses target capsid proteins, transduced cells, and gene therapy products, ultimately resulting in loss of enzyme activity. Inherent differences in male versus female immune response can significantly impact AAV gene transfer. We aim to investigate sex differences in the immune response to AAV gene therapies in mice with mucopolysaccharidosis IVA (MPS IVA). MPS IVA mice, treated with different AAV vectors expressing human N-acetylgalactosamine 6-sulfate sulfatase (GALNS), demonstrated a more robust antibody response in female mice resulting in subsequent decreased GALNS enzyme activity and less therapeutic efficacy in tissue pathology relative to male mice. Under thyroxine-binding globulin promoter, neutralizing antibody titers in female mice were approximately 4.6-fold higher than in male mice, with GALNS enzyme activity levels approximately 6.8-fold lower. Overall, male mice treated with AAV-based gene therapy showed pathological improvement in the femur and tibial growth plates, ligaments, and articular cartilage as determined by contrasting differences in pathology scores compared to females. Cardiac histology revealed a failure to normalize vacuolation in females, in contrast, to complete correction in male mice. These findings promote the need for further determination of sex-based differences in response to AAV-mediated gene therapy related to developing treatments for MPS IVA.

Sexually dimorphic patterns in electroencephalography power spectrum and autism-related behaviors in a rat model of fragile X syndrome.

Fragile X syndrome (FXS), a neurodevelopmental disorder with autistic features, is caused by the loss of the fragile X mental retardation protein. Sex-specific differences in the clinical profile have been observed in FXS patients, but few studies have directly compared males and females in rodent models of FXS. To address this, we performed electroencephalography (EEG) recordings and a battery of autism-related behavioral tasks on juvenile and young adult Fmr1 knockout (KO) rats. EEG analysis demonstrated that compared to wild-type, male Fmr1 KO rats showed an increase in gamma frequency band power in the frontal cortex during the sleep-like immobile state, and both male and female KO rats failed to show an increase in delta frequency power in the sleep-like state, as observed in wild-type rats. Previous studies of EEG profiles in FXS subjects also reported abnormally increased gamma frequency band power, highlighting this parameter as a potential translatable biomarker. Both male and female Fmr1 KO rats displayed reduced exploratory behaviors in the center zone of the open field test, and increased distance travelled in an analysis of 24-h home cage activity, an effect that was more prominent during the nocturnal phase. Reduced wins against wild-type opponents in the tube test of social dominance was seen in both sexes. In contrast, increased repetitive behaviors in the wood chew test was observed in male but not female KO rats, while increased freezing in a fear conditioning test was observed only in the female KO rats. Our findings highlight sex differences between male and female Fmr1 KO rats, and indicate that the rat model of FXS could be a useful tool for the development of new therapeutics for treating this debilitating neurodevelopmental disorder.

New gorilla adenovirus vaccine vectors induce potent immune responses and protection in a mouse malaria model.

BACKGROUND: A DNA-human Ad5 (HuAd5) prime-boost malaria vaccine has been shown to protect volunteers against a controlled human malaria infection. The potency of this vaccine, however, appeared to be affected by the presence of pre-existing immunity against the HuAd5 vector. Since HuAd5 seroprevalence is very high in malaria-endemic areas of the world, HuAd5 may not be the most appropriate malaria vaccine vector. This report describes the evaluation of the seroprevalence, immunogenicity and efficacy of three newly identified gorilla adenoviruses, GC44, GC45 and GC46, as potential malaria vaccine vectors. RESULTS: The seroprevalence of GC44, GC45 and GC46 is very low, and the three vectors are not efficiently neutralized by human sera from Kenya and Ghana, two countries where malaria is endemic. In mice, a single administration of GC44, GC45 and GC46 vectors expressing a murine malaria gene, Plasmodium yoelii circumsporozoite protein (PyCSP), induced robust PyCSP-specific T cell and antibody responses that were at least as high as a comparable HuAd5-PyCSP vector. Efficacy studies in a murine malaria model indicated that a prime-boost regimen with DNA-PyCSP and GC-PyCSP vectors can protect mice against a malaria challenge. Moreover, these studies indicated that a DNA-GC46-PyCSP vaccine regimen was significantly more efficacious than a DNA-HuAd5-PyCSP regimen. CONCLUSION: These data suggest that these gorilla-based adenovectors have key performance characteristics for an effective malaria vaccine. The superior performance of GC46 over HuAd5 highlights its potential for clinical development.

Identification of a suppressor mutation that improves the yields of hexon-modified adenovirus vectors.

We have generated hexon-modified adenovirus serotype 5 (Ad5) vectors that are not neutralized by Ad5-specific neutralizing antibodies in mice. These vectors are attractive for the advancement of vaccine products because of their potential for inducing robust antigen-specific immune responses in people with prior exposure to Ad5. However, hexon-modified Ad5 vectors displayed an approximate 10-fold growth defect in complementing cells, making potential vaccine costs unacceptably high. Replacing hypervariable regions (HVRs) 1, 2, 4, and 5 with the equivalent HVRs from Ad43 was sufficient to avoid Ad5 preexisting immunity and retain full vaccine potential. However, the resulting vector displayed the same growth defect as the hexon-modified vector carrying all 9 HVRs from Ad43. The growth defect is likely due to a defect in capsid assembly, since DNA replication and late protein accumulation were normal in these vectors. We determined that the hexon-modified vectors have a 32°C cold-sensitive phenotype and selected revertants that restored vector productivity. Genome sequencing identified a single base change resulting in a threonine-to-methionine amino acid substitution at the position equivalent to residue 342 of the wild-type protein. This mutation has a suppressor phenotype (SP), since cloning it into our Ad5 vector containing all nine hypervariable regions from Ad43, Ad5.H(43m-43), increased yields over the version without the SP mutation. This growth improvement was also shown for an Ad5-based hexon-modified vector that carried the hexon hypervariable regions of Ad48, indicating that the SP mutation may have broad applicability for improving the productivity of different hexon-modified vectors.

In silico prediction and in vivo testing of promoters targeting GABAergic inhibitory neurons.

Impairment of GABAergic inhibitory neuronal function is linked to epilepsy and other neurological and psychiatric disorders. Recombinant adeno-associated virus (rAAV)-based gene therapy targeting GABAergic neurons is a promising treatment for GABA-associated disorders. However, there is a need to develop rAAV-compatible gene-regulatory elements capable of selectively driving expression in GABAergic neurons throughout the brain. Here, we designed several novel GABAergic gene promoters. In silico analyses, including evolutionarily conserved DNA sequence alignments and transcription factor binding site searches among GABAergic neuronal genes, were carried out to reveal novel sequences for use as rAAV-compatible promoters. rAAVs (serotype 9) were injected into the CSF of neonatal mice and into the brain parenchyma of adult mice to assess promoter specificity. In mice injected neonatally, transgene expression was detected in multiple brain regions with very high neuronal specificity and moderate-to-high GABAergic neuronal selectivity. The GABA promoters differed greatly in their levels of expression and, in some brain regions, showed strikingly different patterns of GABAergic neuron transduction. This study is the first report of rAAV vectors that are functional in multiple brain regions using promoters designed by in silico analyses from multiple GABAergic genes. These novel GABA-targeting vectors may be useful tools to advance gene therapy for GABA-associated disorders.

Kinetics and durability of transgene expression after intrastriatal injection of AAV9 vectors.

Understanding the kinetics and durability of AAV-mediated transgene expression in the brain is essential for conducting basic neuroscience studies as well as for developing gene therapy approaches for CNS diseases. Here, we characterize and compare the temporal profile of transgene expression after bilateral injections into the mouse striatum of rAAV9 encoding GFP under the control of either a ubiquitous promoter (CAG), or the neuron-specific human synapsin (hSyn) and CamKII promoters. GFP protein expression with the CAG promoter was highest at 3 weeks, and then decreased to stable levels at 3 and 6 months. Surprisingly, GFP mRNA levels continued to increase from 3 weeks to 3 months, despite GFP protein expression decreasing during this time. GFP protein expression with hSyn increased more slowly, reaching a maximum at 3 months, which was equivalent to protein expression levels from CAG at that time point. Importantly, transgene expression driven by the hSyn promoter at 6 months was not silenced as previously reported, and GFP mRNA was continuing to rise even at the final 6-month time point. Thus, hSyn as a promoter for transgene expression demonstrates long-term durability but may require more time after vector administration to achieve steady-state levels. Because CAG had the highest GFP protein expression in our comparison, which was at 3 weeks post administration, the early kinetics of transgene expression from CAG was examined (1, 2, 5, and 10 days after injection). This analysis showed that GFP protein expression and GFP mRNA increased during the first 3 weeks after administration. Interestingly, vector DNA rapidly decreased 10-fold over the first 3 weeks following injection as it assembled into stable circular episomes and concatemers. Surprisingly, the processing of vector genomes into circular episomes and concatemers was continually dynamic up to 3 months after injection. These results provide novel insight into the dynamic processing of vector genomes and promoter-specific temporal patterns of transgene expression in the brain.

Gene therapy using an ortholog of human fragile X mental retardation protein partially rescues behavioral abnormalities and EEG activity.

Fragile X syndrome (FXS), a neurodevelopmental disorder with no known cure, is caused by a lack of expression of the fragile X mental retardation protein (FMRP). As a single-gene disorder, FXS is an excellent candidate for viral-vector-based gene therapy, although that is complicated by the existence of multiple isoforms of FMRP, whose individual cellular functions are unknown. We studied the effects of rat and mouse orthologs of human isoform 17, a major expressed isoform of FMRP. Injection of neonatal Fmr1 knockout rats and mice with adeno-associated viral vectors (AAV9 serotype) under the control of an MeCP2 mini-promoter resulted in widespread distribution of the FMRP transgenes throughout the telencephalon and diencephalon. Transgene expression occurred mainly in non-GABAergic neurons, with little expression in glia. Early postnatal treatment resulted in partial rescue of the Fmr1 KO rat phenotype, including improved social dominance in treated Fmr1 KO females and partial rescue of locomotor activity in males. Electro-encephalogram (EEG) recordings showed correction of abnormal slow-wave activity during the sleep-like state in male Fmr1 KO rats. These findings support the use of AAV-based gene therapy as a treatment for FXS and specifically demonstrate the potential therapeutic benefit of human FMRP isoform 17 orthologs.

Identification and localization of minimal MHC-restricted CD8+ T cell epitopes within the Plasmodium falciparum AMA1 protein.

BACKGROUND: Plasmodium falciparum apical membrane antigen-1 (AMA1) is a leading malaria vaccine candidate antigen that is expressed by sporozoite, liver and blood stage parasites. Since CD8+ T cell responses have been implicated in protection against pre-erythrocytic stage malaria, this study was designed to identify MHC class I-restricted epitopes within AMA1. METHODS: A recombinant adenovirus serotype 5 vector expressing P. falciparum AMA1 was highly immunogenic when administered to healthy, malaria-naive adult volunteers as determined by IFN-γ ELISpot responses to peptide pools containing overlapping 15-mer peptides spanning full-length AMA1. Computerized algorithms (NetMHC software) were used to predict minimal MHC-restricted 8-10-mer epitope sequences within AMA1 15-mer peptides active in ELISpot. A subset of epitopes was synthesized and tested for induction of CD8+ T cell IFN-γ responses by ELISpot depletion and ICS assays. A 3-dimensional model combining Domains I + II of P. falciparum AMA1 and Domain III of P. vivax AMA1 was used to map these epitopes. RESULTS: Fourteen 8-10-mer epitopes were predicted to bind to HLA supertypes A01 (3 epitopes), A02 (4 epitopes), B08 (2 epitopes) and B44 (5 epitopes). Nine of the 14 predicted epitopes were recognized in ELISpot or ELISpot and ICS assays by one or more volunteers. Depletion of T cell subsets confirmed that these epitopes were CD8+ T cell-dependent. A mixture of the 14 minimal epitopes was capable of recalling CD8+ T cell IFN-γ responses from PBMC of immunized volunteers. Thirteen of the 14 predicted epitopes were polymorphic and the majority localized to the more conserved front surface of the AMA1 model structure. CONCLUSIONS: This study predicted 14 and confirmed nine MHC class I-restricted CD8+ T cell epitopes on AMA1 recognized in the context of seven HLA alleles. These HLA alleles belong to four HLA supertypes that have a phenotypic frequency between 23% - 100% in different human populations.

Molecular vaccines for malaria.

The basic premise of vaccination is the triggering of host immune responses leading to the induction of adaptive immunity having sufficient magnitude and duration to provide long term protection. This has been achieved by many licensed vaccines, the majority based on attenuated or inactivated organisms, although often the protective antigens and underlying molecular mechanisms have not been identified. However, this traditional approach has not led to the development of a licensed vaccine for malaria or for several other devastating infectious diseases. Recently, substantial efforts have been focused on applying rational molecular design principles toward the development of novel vaccines for these refractory pathogens. In this review, we discuss the molecular aspects of antigen design, adjuvant advancement and the development of vaccine delivery systems as they are being applied to malaria vaccines.

Liver-Targeted AAV8 Gene Therapy Ameliorates Skeletal and Cardiovascular Pathology in a Mucopolysaccharidosis IVA Murine Model.

Mucopolysaccharidosis type IVA (MPS IVA) is due to the deficiency of GALNS (N-acetylgalactosamine 6-sulfate sulfatase) and is characterized by systemic skeletal dysplasia. We have evaluated adeno-associated virus 8 (AAV8) vectors expressing different forms of human GALNS under a liver-specific promoter. The vectors were delivered intravenously into 4-week-old MPS IVA knockout (KO) and immune tolerant (MTOL) mice at a dose of 5 × 1013 genome copies (GC)/kg. These mice were monitored for 12 weeks post-injection. GALNS enzyme activity was elevated significantly in plasma of all treated mice at 2 weeks post-injection. The activity observed was 4- to 19-fold higher than that in wild-type mice and was maintained throughout the monitoring period. Treatment with AAV vectors resulted in a reduction of keratan sulfate (KS) levels in plasma to normal levels 2 weeks post-injection, which were maintained until necropsy. Both vectors reduced the storage in articular cartilage, ligaments, and meniscus surrounding articular cartilage and growth plate region as well as heart muscle and valves. Our results suggest that the continuous presence of high levels of circulating enzyme increases the penetration into bone and heart and reduces the KS level, thereby improving storage in these regions. The current data support a strategy for developing a novel treatment to address the bone and heart disease in MPS IVA using AAV gene therapy.

Persistent expression of PEDF in the eye using high-capacity adenovectors.

Ocular neovascularization, the growth of abnormal blood vessels in the eye, is a factor shared by the most common blinding diseases in developed countries. Pigment epithelium-derived factor (PEDF) is a potent antiangiogenic and neuroprotective protein that is normally produced in the eye. When delivered via an adenovector, PEDF can block the growth of new blood vessels and trigger the selective regression of abnormal vessels in animal models of ocular disease. Because of the absence of adenoviral genes, high-capacity (HC) adenovectors offer the potential for persistent transgene expression and enhanced tolerability. We have assessed the durability of PEDF expression and the induction of ocular inflammation following delivery of a PEDF-expressing HC adenovector compared to earlier generation vectors. The HC vector mediated prolonged PEDF expression in tissue-cultured pigmented epithelial cells and when delivered by intravitreal injection into the mouse eye. Delivery of first-generation adenovectors resulted in a dose-dependent increase in cytokine/chemokine gene expression, which correlated with the infiltration of inflammatory cells in the eye. In comparison, the levels of inflammatory gene expression and the intraocular infiltrate were substantially reduced following delivery of the HC vector. These results support the development of the HC adenovector gene delivery system for ocular disease.

Protection against P. aeruginosa with an adenovirus vector containing an OprF epitope in the capsid.

Pseudomonas aeruginosa is an important opportunistic pathogen that can cause chronic and often life-threatening infections of the respiratory tract, particularly in individuals with cystic fibrosis (CF). Because infections with P. aeruginosa remain the major cause of the high morbidity and mortality of CF, a vaccine against P. aeruginosa would be very useful for preventing this disorder. The outer membrane protein F (OprF) of P. aeruginosa is a promising vaccine candidate and various B cell epitopes within OprF have been identified. Given that adenovirus (Ad) vectors have strong immunogenic potential and can function as adjuvants for genetic vaccines, the present study evaluates the immunogenic and protective properties of a novel replication-deficient Ad vector in which the Ad hexon protein was modified to include a 14-amino acid epitope of P. aeruginosa OprF (Epi8) in loop 1 of the hypervariable region 5 of the hexon (AdZ.Epi8). Immunization of C57BL/6 mice with AdZ.Epi8 resulted in detectable serum anti-P. aeruginosa and anti-OprF humoral responses. These responses were haplotype dependent, with higher serum anti-OprF titers in CBA mice than in BALB/c or C57BL/6 mice. AdZ.Epi8 induced Epi8-specific IFN-gamma-positive CD4 and CD8 T cell responses and resulted in protection against a lethal pulmonary challenge with agar-encapsulated P. aeruginosa. Importantly, repeated administration of AdZ.Epi8 resulted in boosting of the anti-OprF humoral and anti-Epi8 cellular response, whereas no boosting effect was present in the response against the transgene beta-galactosidase. These observations suggest that Ad vectors expressing pathogen epitopes in their capsid will protect against an extracellular pathogen and will allow boosting of the epitope-specific humoral response with repeated administration, a strategy that should prove useful in developing Ad vectors as vaccines where humoral immunity will be protective.

Profiling the Targets of Protective CD8+ T Cell Responses to Infection.

T cells are critical effectors of host immunity that target intracellular pathogens, such as the causative agents of HIV, tuberculosis, and malaria. The development of vaccines that induce effective cell-mediated immunity against such pathogens has proved challenging; for tuberculosis and malaria, many of the antigens targeted by protective T cells are not known. Here, we report a novel approach for screening large numbers of antigens as potential targets of T cells. Malaria provides an excellent model to test this antigen discovery platform because T cells are critical mediators of protection following immunization with live sporozoite vaccines and the specific antigen targets are unknown. We generated an adenovirus array by cloning 312 highly expressed pre-erythrocytic Plasmodium yoelii antigens into adenovirus vectors using high-throughput methodologies. The array was screened to identify antigen-specific CD8+ T cells induced by a live sporozoite vaccine regimen known to provide high levels of sterile protection mediated by CD8+ T cells. We identified 69 antigens that were targeted by CD8+ T cells induced by this vaccine regimen. The antigen that recalled the highest frequency of CD8+ T cells, PY02605, induced protective responses in mice, demonstrating proof of principle for this approach in identifying antigens for vaccine development.

Repeated administration of adenovector in the eye results in efficient gene delivery.

PURPOSE: To determine whether repeat administration of an adenovector (Ad) into the eye results in efficient gene delivery and to test whether transgenes can be expressed from an adenovector expression system in the presence of preexisting, neutralizing anti-Ad antibodies. METHODS: To assess the efficiency of repeated gene delivery of an adenovector expression system, C57Bl/6 mice received one, two, or three injections (intravitreal [IVT] or periocular [PO]) of AdNull.11D (empty cassette) at 2-week intervals, followed by a single AdLuciferase (AdL.11D) IVT or PO injection. Mice were killed approximately 24 hours after AdL.11D injection and the eyes were enucleated and stored until assayed. Serum samples were also analyzed to determine whether repeated IVT or PO injections lead to induction of neutralizing antibodies directed against an adenovector delivery system. To determine whether preexisting neutralizing anti-Ad antibodies would block transgene expression, mice were preimmunized with one, two, or three intramuscular (IM) injection(s) of AdNull.11D (1 x 10(9) particle units [pu]). Fourteen days later, when systemic anti-Ad antibody titers were expected to exist, mice were given a single AdL.11D injection (IVT or PO) and killed, and the eyes and serum collected. RESULTS: These studies show that multiple injections at 2-week intervals with adenovectors (IM, IVT, or PO) did not prevent transgene expression in the eye. Moreover, measurement of neutralizing anti-Ad antibody titers revealed that measurable anti-Ad antibody titers in mice did not ablate transgene expression. CONCLUSIONS: These studies suggest that transgene expression after repeated adenovector administration into the eye is feasible and repeated injections, whether given IVT or PO, do not lead to an immediate increase in neutralizing anti-Ad antibody titers. Moreover, preimmunization of mice by systemic exposure to adenovector, does not block transgene expression in the eye. These studies indicate that repeat administration of adenovectors (IVT and PO) into the eye can be considered in designing future clinical trials and that the pre-existence of neutralizing anti-Ad antibodies probably does not mitigate activity.

Adenoviruses isolated from wild gorillas are closely related to human species C viruses.

We have isolated and cultured three distinct adenoviruses from wild gorillas. Phylogenetic analysis grouped the viruses with human adenovirus species C based on DNA polymerase, hexon, and E4ORF6 genes. The three wild gorilla adenoviruses clustered with the other species C captive gorilla adenoviruses, forming a branch separate from human and chimpanzee/bonobo adenoviruses. Animal sera to the three newly isolated viruses did not cross-neutralize, demonstrating serological distinctiveness. The human adenovirus 5 fiber knob blocked infection, suggesting use of the Coxsackie and Adenovirus Receptor. These viruses may provide viral vectors with properties distinct from chimpanzee adenovirus and human adenovirus vectors.

A glycolipid adjuvant, 7DW8-5, enhances CD8+ T cell responses induced by an adenovirus-vectored malaria vaccine in non-human primates.

A key strategy to a successful vaccine against malaria is to identify and develop new adjuvants that can enhance T-cell responses and improve protective immunity. Upon co-administration with a rodent malaria vaccine in mice, 7DW8-5, a recently identified novel analog of α-galactosylceramide (α-GalCer), enhances the level of malaria-specific protective immune responses more strongly than the parent compound. In this study, we sought to determine whether 7DW8-5 could provide a similar potent adjuvant effect on a candidate human malaria vaccine in the more relevant non-human primate (NHP) model, prior to committing to clinical development. The candidate human malaria vaccine, AdPfCA (NMRC-M3V-Ad-PfCA), consists of two non-replicating recombinant adenoviral (Ad) vectors, one expressing the circumsporozoite protein (CSP) and another expressing the apical membrane antigen-1 (AMA1) of Plasmodium falciparum. In several phase 1 clinical trials, AdPfCA was well tolerated and demonstrated immunogenicity for both humoral and cell-mediated responses. In the study described herein, 25 rhesus macaques received prime and boost intramuscular (IM) immunizations of AdPfCA alone or with an ascending dose of 7DW8-5. Our results indicate that 7DW8-5 is safe and well-tolerated and provides a significant enhancement (up to 9-fold) in malaria-specific CD8+ T-cell responses after both priming and boosting phases, supporting further clinical development.

Human adenovirus 5-vectored Plasmodium falciparum NMRC-M3V-Ad-PfCA vaccine encoding CSP and AMA1 is safe, well-tolerated and immunogenic but does not protect against controlled human malaria infection.

BACKGROUND: In a prior study, a DNA prime / adenovirus boost vaccine (DNA/Ad) expressing P. falciparum circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1) (NMRC-M3V-D/Ad-PfCA Vaccine) induced 27% protection against controlled human malaria infection (CHMI). To investigate the contribution of DNA priming, we tested the efficacy of adenovirus vaccine alone (NMRC-M3V-Ad-PfCA ) in a Phase 1 clinical trial. METHODOLOGY/PRINCIPAL FINDINGS: The regimen was a single intramuscular injection with two non-replicating human serotype 5 adenovectors encoding CSP and AMA1, respectively. One x 10 (10) particle units of each construct were combined prior to administration. The regimen was safe and well-tolerated. Four weeks later, 18 study subjects received P. falciparum CHMI administered by mosquito bite. None were fully protected although one showed delayed onset of parasitemia. Antibody responses were low, with geometric mean CSP ELISA titer of 381 (range<50-1626) and AMA1 ELISA of 4.95 µg/mL (range 0.2-38). Summed ex vivo IFN-γ ELISpot responses to overlapping peptides were robust, with geometric mean spot forming cells/million peripheral blood mononuclear cells [sfc/m] for CSP of 273 (range 38-2550) and for AMA1 of 1303 (range 435-4594). CD4+ and CD8+ T cell IFN-γ responses to CSP were positive by flow cytometry in 25% and 56% of the research subjects, respectively, and to AMA1 in 94% and 100%, respectively. SIGNIFICANCE: In contrast to DNA/Ad, Ad alone did not protect against CHMI despite inducing broad, cell-mediated immunity, indicating that DNA priming is required for protection by the adenovirus-vectored vaccine. ClinicalTrials.gov Identifier: NCT00392015.

DNA prime/Adenovirus boost malaria vaccine encoding P. falciparum CSP and AMA1 induces sterile protection associated with cell-mediated immunity.

BACKGROUND: Gene-based vaccination using prime/boost regimens protects animals and humans against malaria, inducing cell-mediated responses that in animal models target liver stage malaria parasites. We tested a DNA prime/adenovirus boost malaria vaccine in a Phase 1 clinical trial with controlled human malaria infection. METHODOLOGY/PRINCIPAL FINDINGS: The vaccine regimen was three monthly doses of two DNA plasmids (DNA) followed four months later by a single boost with two non-replicating human serotype 5 adenovirus vectors (Ad). The constructs encoded genes expressing P. falciparum circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1). The regimen was safe and well-tolerated, with mostly mild adverse events that occurred at the site of injection. Only one AE (diarrhea), possibly related to immunization, was severe (Grade 3), preventing daily activities. Four weeks after the Ad boost, 15 study subjects were challenged with P. falciparum sporozoites by mosquito bite, and four (27%) were sterilely protected. Antibody responses by ELISA rose after Ad boost but were low (CSP geometric mean titer 210, range 44-817; AMA1 geometric mean micrograms/milliliter 11.9, range 1.5-102) and were not associated with protection. Ex vivo IFN-γ ELISpot responses after Ad boost were modest (CSP geometric mean spot forming cells/million peripheral blood mononuclear cells 86, range 13-408; AMA1 348, range 88-1270) and were highest in three protected subjects. ELISpot responses to AMA1 were significantly associated with protection (p = 0.019). Flow cytometry identified predominant IFN-γ mono-secreting CD8+ T cell responses in three protected subjects. No subjects with high pre-existing anti-Ad5 neutralizing antibodies were protected but the association was not statistically significant. SIGNIFICANCE: The DNA/Ad regimen provided the highest sterile immunity achieved against malaria following immunization with a gene-based subunit vaccine (27%). Protection was associated with cell-mediated immunity to AMA1, with CSP probably contributing. Substituting a low seroprevalence vector for Ad5 and supplementing CSP/AMA1 with additional antigens may improve protection. TRIAL REGISTRATION: ClinicalTrials.govNCT00870987.