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Human pluripotent stem cells have the potential to differentiate into various cell types including skeletal muscles (SkM), and they are applied to regenerative medicine or in vitro modelling for intractable diseases. A simple differentiation method is required for SkM cells to accelerate neuromuscular disease studies. Here, we established a simple method to convert human pluripotent stem cells into SkM cells by using temperature-sensitive Sendai virus (SeV) vector encoding myoblast determination protein 1 (SeV-Myod1), a myogenic master transcription factor. SeV-Myod1 treatment converted human embryonic stem cells (ESCs) into SkM cells, which expressed SkM markers including myosin heavy chain (MHC). We then removed the SeV vector by temporal treatment at a high temperature of 38℃, which also accelerated mesodermal differentiation, and found that SkM cells exhibited fibre-like morphology. Finally, after removal of the residual human ESCs by pluripotent stem cell-targeting delivery of cytotoxic compound, we generated SkM cells with 80% MHC positivity and responsiveness to electrical stimulation. This simple method for myogenic differentiation was applicable to human-induced pluripotent stem cells and will be beneficial for investigations of disease mechanisms and drug discovery in the future., 温度感受性センダイウイルスベクターを用いて ヒトES細胞／iPS細胞から骨格筋細胞を簡便に作製する技術開発 --神経筋疾患病態モデル構築と創薬研究への利用--. 京都大学プレスリリース. 2021-09-13.

Effective T cell induction is an important strategy in HIV-vaccine development. However, it has been indicated that vaccine-induced HIV-specific CD4

Human motor neurons are important materials for the research of the pathogenesis and drug discovery of motor neuron diseases. Various methods to generate motor neurons (MNs) from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) by the addition of signaling molecules have been reported. However, they require multiple steps and complicated processes. Here we describe an approach for generating human MNs from ESCs/iPSCs using a single Sendai virus vector encoding three transcription factors-Lhx3, Ngn2, and Isl1. This approach enabled us to generate MNs in one step, adding Sendai virus vector in culture medium. This simple method significantly reduces the efforts to generate MNs, and it provides a useful tool for motor neuron disease research.

Induction of antibodies targeting viral glycoproteins is a key for the development of a vaccine against enveloped virus infection. Glycoproteins on the virion exhibiting native multimer structure may be a good immunogen to present antibody epitopes, but it is often difficult to prepare immunogenic inactivated virions. Preparation of soluble glycoprotein multimers has been attempted, while virus-like particles carrying target glycoproteins can be a more immunogenic antigen. In the present study, a target glycoprotein-embedded Sendai virus (SeV) particle was developed for induction of anti-virus antibodies. We constructed a chimeric antigen, HIV-1 EnvF, consisting of HIV-1 Env ectodomain and SeV F transmembrane-cytoplasmic domain, which was shown to be efficiently incorporated into the SeV virion. EnvF was recognized by anti-HIV-1 broadly-neutralizing monoclonal antibodies (bnAbs) including 35O22 that targets an Env trimer-dependent epitope. Analysis revealed that HIV-1

Adult hearts have limited regenerative capacity. Hence, after acute myocardial infarction (MI), dead myocardial tissues are digested by immune cells and replaced by fibrosis, leading to ventricular remodeling and heart failure at the chronic stage. Direct reprogramming of the cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) with cardiac transcription factors, including Gata4, Mef2c, and Tbx5 (GMT), may have significant potential for cardiac repair. Sendai virus (SeV) vectors expressing GMT have been reported to reprogram the mouse cardiac fibroblasts into iCMs without any risk of insertional mutagenesis. In vivo reprogramming improved the cardiac function after acute MI in immunodeficient mice. However, it is unknown whether the newly generated iCMs could exist in infarct hearts for a prolonged period and SeV-GMT can improve cardiac function after MI at the chronic stage in immunocompetent mice. Here, we show that SeV vectors efficiently infect CFs in vivo and reprogram them into iCMs, which existed for at least four weeks after MI, in fibroblast-linage tracing mice. Moreover, SeV-GMT improved cardiac function and reduced fibrosis and collagen I expression at 12 weeks after MI in immunocompetent mice. Thus, direct cardiac reprogramming with SeV vectors could be a promising therapy for MI.

Because both AIDS and tuberculosis are serious health threats in middle/low-income countries, development of a dual vaccine against them would be highly beneficial. To approach the goal, here we first assessed a urease-deficient bacillus Calmette-Guérin (BCG) for improvement of immunogenicity against both Mycobacterium tuberculosis and SIV. Second, we demonstrated the usefulness of Asian-origin cynomolgus monkeys for development of a preclinical AIDS vaccine by direct comparison with Indian rhesus macaques as the only validated hosts that identically mirror the outcomes of clinical trials, since the availability of Indian rhesus macaques is limited in countries other than the United States. Finally, we report the protective effect of a vaccination regimen comprising BCG, the highly attenuated vaccinia virus LC16m8Δ strain, and nontransmissible Sendai virus as safe vectors expressing SIV genes using repeated mucosal challenge with highly pathogenic SIVmac251. Identification of CD8+ T cells as a protective immunity suggests a future direction of AIDS vaccine development., Toward development of a dual vaccine for human immunodeficiency virus type 1 (HIV-1) and tuberculosis infections, we developed a urease-deficient bacillus Calmette-Guérin (BCG) strain Tokyo172 (BCGΔurease) to enhance its immunogenicity. BCGΔurease expressing a simian immunodeficiency virus (SIV) Gag induced BCG antigen-specific CD4+ and CD8+ T cells more efficiently and more Gag-specific CD8+ T cells. We evaluated its protective efficacy against SIV infection in cynomolgus monkeys of Asian origin, shown to be as susceptible to infection with SIVmac251 as Indian rhesus macaques. Priming with recombinant BCG (rBCG) expressing SIV genes was followed by a boost with SIV gene-expressing LC16m8Δ vaccinia virus and a second boost with SIV Env-expressing Sendai virus. Eight weeks after the second boost, monkeys were repeatedly challenged with a low dose of SIVmac251 intrarectally. Two animals out of 6 vaccinees were protected, whereas all 7 control animals were infected without any early viral controls. In one vaccinated animal, which had the most potent CD8+ T cells in an in vitro suppression activity (ISA) assay of SIVmac239 replication, plasma viremia was undetectable throughout the follow-up period. Protection was confirmed by the lack of anamnestic antibody responses and detectable cell-associated provirus in various organs. Another monkey with a high ISA acquired a small amount of SIV, but it later became suppressed below the detection limit. Moreover, the ISA score correlated with SIV acquisition. On the other hand, any parameter relating anti-Env antibody was not correlated with the protection. IMPORTANCE Because both AIDS and tuberculosis are serious health threats in middle/low-income countries, development of a dual vaccine against them would be highly beneficial. To approach the goal, here we first assessed a urease-deficient bacillus Calmette-Guérin (BCG) for improvement of immunogenicity against both Mycobacterium tuberculosis and SIV. Second, we demonstrated the usefulness of Asian-origin cynomolgus monkeys for development of a preclinical AIDS vaccine by direct comparison with Indian rhesus macaques as the only validated hosts that identically mirror the outcomes of clinical trials, since the availability of Indian rhesus macaques is limited in countries other than the United States. Finally, we report the protective effect of a vaccination regimen comprising BCG, the highly attenuated vaccinia virus LC16m8Δ strain, and nontransmissible Sendai virus as safe vectors expressing SIV genes using repeated mucosal challenge with highly pathogenic SIVmac251. Identification of CD8+ T cells as a protective immunity suggests a future direction of AIDS vaccine development.

Human motor neurons are important materials for the research of the pathogenesis and drug discovery of motor neuron diseases. Various methods to generate motor neurons (MNs) from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) by the addition of signaling molecules have been reported. However, they require multiple steps and complicated processes. Here we describe an approach for generating human MNs from ESCs/iPSCs using a single Sendai virus vector encoding three transcription factors-Lhx3, Ngn2, and Isl1. This approach enabled us to generate MNs in one step, adding Sendai virus vector in culture medium. This simple method significantly reduces the efforts to generate MNs, and it provides a useful tool for motor neuron disease research.

Anti-retroviral therapy (ART) can inhibit HIV proliferation but not achieve virus eradication from HIV-infected individuals. Under ART-based HIV control, virus-specific CD8+ T-cell responses are often reduced. Here, we investigated the impact of therapeutic vaccination inducing virus-specific CD8+ T-cell responses under ART on viral control in a macaque AIDS model. Twelve rhesus macaques received ART from week 12 to 32 after simian immunodeficiency virus (SIV) infection. Six of them were vaccinated with Sendai virus vectors expressing SIV Gag and Vif at weeks 26 and 32, and Gag/Vif-specific CD8+ T-cell responses were enhanced and became predominant. All macaques controlled viremia during ART but showed viremia rebound after ART cessation. Analysis of in vitro CD8+ cell ability to suppress replication of autologous lymphocytes-derived SIVs found augmentation of anti-SIV efficacy of CD8+ cells after vaccination. In the vaccinated animals, the anti-SIV efficacy of CD8+ cells at week 34 was correlated positively with Gag-specific CD8+ T-cell frequencies and inversely with rebound viral loads at week 34. These results indicate that Gag-specific CD8+ T-cell induction by therapeutic vaccination can augment anti-virus efficacy of CD8+ cells, which may be insufficient for functional cure but contribute to more stable viral control under ART.

Optimization of immunogen is crucial for induction of effective T-cell responses in the development of a human immunodeficiency virus (HIV) vaccine. Conventional T-cell-based vaccines have been designed to induce virus-specific CD4+ T as well as CD8+ T cells. However, it has been indicated that induction of HIV-specific CD4+ T cells, preferential targets for HIV infection, by vaccination may be detrimental and accelerate viral replication after HIV exposure. In the present study, we present a novel immunogen to selectively induce CD8+ T cells but not CD4+ T cells targeting viral antigens. The immunogen, CaV11, was constructed by tandem connection of overlapping 11-mer peptides spanning simian immunodeficiency virus (SIV) Gag capsid (CA) and Vif. Prime-boost immunization with DNA and Sendai virus (SeV) vectors expressing CaV11 efficiently induced Gag/Vif-specific CD8+ T-cell responses with inefficient Gag/Vif-specific CD4+ T-cell induction in rhesus macaques (n = 6). None of the macaques exhibited the enhancement of acute viral replication after an intravenous high-dose SIV challenge, which was observed in those immunized with DNA and SeV expressing the whole Gag protein in our previous study. Set point viral control postinfection was associated with SeV-specific CD4+ T-cell responses postimmunization, suggesting contribution of SeV-specific helper responses to effective Gag/Vif-specific CD8+ T-cell induction by vaccination. This immunogen design could be a promising method for selective induction of effective anti-HIV CD8+ T-cell responses.IMPORTANCE Induction of effective CD8+ T-cell responses is an important HIV vaccine strategy. Several promising vaccine delivery tools have been developed, and immunogen optimization is now crucial for effective T-cell induction. Conventional immunogens have been designed to induce virus-specific CD4+ T cells as well as CD8+ T cells, but induction of virus-specific CD4+ T cells that are preferential targets for HIV infection could enhance acute HIV proliferation. Here, we designed a novel immunogen to induce HIV-specific CD8+ T cells without HIV-specific CD4+ T-cell induction but with non-HIV antigen-specific CD4+ T-cell help. Our analysis in a macaque AIDS model showed that our immunogen can efficiently elicit effective CD8+ T but not CD4+ T cells targeting viral antigens, resulting in no enhancement of acute viral replication after virus exposure. This immunogen design, also applicable for other currently developed immunogens, could be a promising method for selective induction of effective anti-HIV CD8+ T-cell responses.

Anti-retroviral therapy (ART) can inhibit HIV proliferation but not achieve virus eradication from HIV-infected individuals. Under ART-based HIV control, virus-specific CD8+ T-cell responses are often reduced. Here, we investigated the impact of therapeutic vaccination inducing virus-specific CD8+ T-cell responses under ART on viral control in a macaque AIDS model. Twelve rhesus macaques received ART from week 12 to 32 after simian immunodeficiency virus (SIV) infection. Six of them were vaccinated with Sendai virus vectors expressing SIV Gag and Vif at weeks 26 and 32, and Gag/Vif-specific CD8+ T-cell responses were enhanced and became predominant. All macaques controlled viremia during ART but showed viremia rebound after ART cessation. Analysis of in vitro CD8+ cell ability to suppress replication of autologous lymphocytes-derived SIVs found augmentation of anti-SIV efficacy of CD8+ cells after vaccination. In the vaccinated animals, the anti-SIV efficacy of CD8+ cells at week 34 was correlated positively with Gag-specific CD8+ T-cell frequencies and inversely with rebound viral loads at week 34. These results indicate that Gag-specific CD8+ T-cell induction by therapeutic vaccination can augment anti-virus efficacy of CD8+ cells, which may be insufficient for functional cure but contribute to more stable viral control under ART.

Optimization of immunogen is crucial for induction of effective T-cell responses in the development of a human immunodeficiency virus (HIV) vaccine. Conventional T-cell-based vaccines have been designed to induce virus-specific CD4(+) T as well as CD8(+) T cells. However, it has been indicated that induction of HIV-specific CD4(+) T cells, preferential targets for HIV infection, by vaccination may be detrimental and accelerate viral replication after HIV exposure. In the present study, we present a novel immunogen to selectively induce CD8(+) T cells but not CD4(+) T cells targeting viral antigens. The immunogen, CaV11, was constructed by tandem connection of overlapping 11-mer peptides spanning simian immunodeficiency virus (SIV) Gag capsid (CA) and Vif. Prime-boost immunization with DNA and Sendai virus (SeV) vectors expressing CaV11 efficiently induced Gag/Vif-specific CD8(+) T-cell responses with inefficient Gag/Vif-specific CD4(+) T-cell induction in rhesus macaques (n = 6). None of the macaques exhibited the enhancement of acute viral replication after an intravenous high-dose SIV challenge, which was observed in those immunized with DNA and SeV expressing the whole Gag protein in our previous study. Set point viral control postinfection was associated with SeV-specific CD4(+) T-cell responses postimmunization, suggesting contribution of SeV-specific helper responses to effective Gag/Vif-specific CD8(+) T-cell induction by vaccination. This immunogen design could be a promising method for selective induction of effective anti-HIV CD8(+) T-cell responses. IMPORTANCE Induction of effective CD8(+) T-cell responses is an important HIV vaccine strategy. Several promising vaccine delivery tools have been developed, and immunogen optimization is now crucial for effective T-cell induction. Conventional immunogens have been designed to induce virus-specific CD4(+) T cells as well as CD8(+) T cells, but induction of virus-specific CD4(+) T cells that are preferential targets for HIV infection could enhance acute HIV proliferation. Here, we designed a novel immunogen to induce HIV-specific CD8(+) T cells without HIV-specific CD4(+) T-cell induction but with non-HIV antigen-specific CD4(+) T-cell help. Our analysis in a macaque AIDS model showed that our immunogen can efficiently elicit effective CD8(+) T but not CD4(+) T cells targeting viral antigens, resulting in no enhancement of acute viral replication after virus exposure. This immunogen design, also applicable for other currently developed immunogens, could be a promising method for selective induction of effective anti-HIV CD8(+) T-cell responses.

Joint bleeding and resultant arthropathy are major determinants of quality of life in haemophilia patients. We previously developed a mesenchymal stromal cell (MSC)-based treatment approach for haemophilic arthropathy in a mouse model of haemophilia A. Here, we evaluated the long-term safety of intra-articular injection of lentivirally transduced autologous MSCs in non-human primates. Autologous bone-marrow-derived MSCs transduced with a lentiviral vector expressing coagulation factor VIII (FVIII) were injected into the left knee joint of cynomolgus monkeys. We first conducted codon optimization to increase FVIII production in the cells. Lentiviral transduction of autologous MSCs resulted in a significant increase of FVIII in the culture supernatant before transplantation. We did not find any tumour generation around the knee structure at 11-16 months after injection by magnetic resonance imaging. The proviral sequence of the simian immunodeficiency virus lentiviral vector was not detected in the heart, lungs, spleen, liver, testis, or bone marrow by real-time quantitative PCR. We confirmed the long-term safety of intra-articular injection of transduced MSCs in a non-human primate. The procedure may be an attractive therapeutic approach for joint diseases in haemophilia patients.

In an earlier study, a novel Sendai virus-vectored anti-tuberculosis vaccine encoding Ag85A and Ag85B (SeV85AB) was constructed and shown to elicit antigen-specific T cell responses and protection against Mycobacterium tuberculosis (Mtb) infection in a murine model. In this study, we evaluate whether the immune responses induced by this novel vaccine might be elevated by a recombinant DNA vaccine expressing the same antigen in a heterologous prime-boost vaccination strategy. The results showed that both SeV85AB prime-DNA boost (SeV85AB-DNA) and DNA prime-SeV85AB boost (DNA-SeV85AB) vaccination strategies significantly enhanced the antigen-specific T cell responses induced by the separate vaccines. The SeV85AB-DNA immunization regimen induced higher levels of recall T cell responses after Mtb infection and conferred better immune protection compared with DNA-SeV85AB or a single immunization. Collectively, our study lends strong evidence that a DNA vaccine boost might be included in a novel SeV85AB immunization strategy designed to enhance the immune protection against Mtb. KEY MESSAGES: A heterologous prime-boost regimen with a novel recombinant SeV85AB and a DNA vaccine increase the T cell responses above those from a single vaccine. The heterologous prime-boost regimen provided protection against Mtb infection. The DNA vaccine might be included in a novel SeV85AB immunization strategy designed to enhance the immune protection against Mtb.

Background. We report the first-in-human safety and immunogenicity assessment of a prototype intranasally administered, replication-competent Sendai virus (SeV)–vectored, human immunodeficiency virus type 1 (HIV-1) vaccine. Methods. Sixty-five HIV-1–uninfected adults in Kenya, Rwanda, and the United Kingdom were assigned to receive 1 of 4 prime-boost regimens (administered at 0 and 4 months, respectively; ratio of vaccine to placebo recipients, 12:4): priming with a lower-dose SeV-Gag given intranasally, followed by boosting with an adenovirus 35–vectored vaccine encoding HIV-1 Gag, reverse transcriptase, integrase, and Nef (Ad35-GRIN) given intramuscularly (SLA); priming with a higher-dose SeV-Gag given intranasally, followed by boosting with Ad35-GRIN given intramuscularly (SHA); priming with Ad35-GRIN given intramuscularly, followed by boosting with a higher-dose SeV-Gag given intranasally (ASH); and priming and boosting with a higher-dose SeV-Gag given intranasally (SHSH). Results. All vaccine regimens were well tolerated. Gag-specific IFN-γ enzyme-linked immunospot–determined response rates and geometric mean responses were higher (96% and 248 spot-forming units, respectively) in groups primed with SeV-Gag and boosted with Ad35-GRIN (SLA and SHA) than those after a single dose of Ad35-GRIN (56% and 54 spot-forming units, respectively) or SeV-Gag (55% and 59 spot-forming units, respectively); responses persisted for ≥8 months after completion of the prime-boost regimen. Functional CD8+ T-cell responses with greater breadth, magnitude, and frequency in a viral inhibition assay were also seen in the SLA and SHA groups after Ad35-GRIN boost, compared with those who received either vaccine alone. SeV-Gag did not boost T-cell counts in the ASH group. In contrast, the highest Gag-specific antibody titers were seen in the ASH group. Mucosal antibody responses were sporadic. Conclusions. SeV-Gag primed functional, durable HIV-specific T-cell responses and boosted antibody responses. The prime-boost sequence appears to determine which arm of the immune response is stimulated. Clinical Trials Registration. {"type":"clinical-trial","attrs":{"text":"NCT01705990","term_id":"NCT01705990"}}NCT01705990.

The kinds of vaccine-induced T cell responses that are beneficial for protection against Mycobacterium tuberculosis (Mtb) infection are not adequately defined. We had shown that a novel Sendai virus vectored vaccine, SeV85AB, was able to enhance immune protection induced by bacille Calmette–Guérin (BCG) in a prime-boost model. However, the profile of T cell responses boosted by SeV85AB was not determined. Herein, we show that the antigen-specific CD4+ and CD8+ T cell responses were both enhanced by the SeV85AB boost after BCG. Different profiles of antigen-specific po T cell subsets were induced in the local (lung) and systemic (spleen) sites. In the spleen, the CD4+ T cell responses that were enhanced by the SeV85AB boost were predominately IL-2 responses, whereas in the lung the greater increases were in IFN-γ- and TNF-α-producing CD4+ T cells; in CD8+ T cells, although IFN-γ was enhanced in both the spleen and lung, only IL-2+TNF-α+CD8+ T subset was boosted in the latter. After a challenge Mtb infection, there were significantly higher levels of recall IL-2 responses in T cells. In contrast, IFN-γ-producing cells were barely boosted by SeV85AB. After Mtb challenge a central memory phenotype of responding CD4+ T cells was a prominent feature in SeV85AB-boosted mice. Thus, our data strongly suggest that the enhanced immune protection induced by SeV85AB boosting was associated with establishment of an increased capacity to recall antigen-specific IL-2-mediated T cell responses and confirms this Sendai virus vector system as a promising candidate to be used in a heterologous prime-boost immunization regimen against TB.

We have recently shown that non-viral gene therapy can stabilise the decline of lung function in patients with cystic fibrosis (CF). However, the effect was modest, and more potent gene transfer agents are still required. Fuson protein (F)/Hemagglutinin/Neuraminidase protein (HN)-pseudotyped lentiviral vectors are more efficient for lung gene transfer than non-viral vectors in preclinical models. In preparation for a first-in-man CF trial using the lentiviral vector, we have undertaken key translational preclinical studies. Regulatory-compliant vectors carrying a range of promoter/enhancer elements were assessed in mice and human air-liquid interface (ALI) cultures to select the lead candidate; cystic fibrosis transmembrane conductance receptor (CFTR) expression and function were assessed in CF models using this lead candidate vector. Toxicity was assessed and 'benchmarked' against the leading non-viral formulation recently used in a Phase IIb clinical trial. Integration site profiles were mapped and transduction efficiency determined to inform clinical trial dose-ranging. The impact of pre-existing and acquired immunity against the vector and vector stability in several clinically relevant delivery devices was assessed. A hybrid promoter hybrid cytosine guanine dinucleotide (CpG)- free CMV enhancer/elongation factor 1 alpha promoter (hCEF) consisting of the elongation factor 1α promoter and the cytomegalovirus enhancer was most efficacious in both murine lungs and human ALI cultures (both at least 2-log orders above background). The efficacy (at least 14% of airway cells transduced), toxicity and integration site profile supports further progression towards clinical trial and pre-existing and acquired immune responses do not interfere with vector efficacy. The lead rSIV.F/HN candidate expresses functional CFTR and the vector retains 90-100% transduction efficiency in clinically relevant delivery devices. The data support the progression of the F/HN-pseudotyped lentiviral vector into a first-in-man CF trial in 2017. ispartof: Thorax vol:72 issue:2 pages:137-147 ispartof: location:England status: published

Accumulating evidence has shown the protective role of CD8+ T cells in vaccine-induced immunity against Mycobacterium tuberculosis (Mtb) despite controversy over their role in natural immunity. However, the current vaccine BCG is unable to induce sufficient CD8+ T cell responses, especially in the lung. Sendai virus, a respiratory RNA virus, is here engineered firstly as a novel recombinant anti-TB vaccine (SeV85AB) that encodes Mtb immuno-dominant antigens, Ag85A and Ag85B. A single mucosal vaccination elicited potent antigen-specific T cell responses and a degree of protection against Mtb challenge similar to the effect of BCG in mice. Depletion of CD8+ T cells abrogated the protective immunity afforded by SeV85AB vaccination. Interestingly, only SeV85AB vaccination induced high levels of lung-resident memory CD8+ T (TRM) cells, and this led to a rapid and strong recall of antigen-specific CD8+ T cell responses against Mtb challenge infection. Furthermore, when used in a BCG prime-SeV85AB boost strategy, SeV85AB vaccine significantly enhanced protection above that seen after BCG vaccination alone. Our findings suggest that CD8+ TRM cells that arise in lungs responding to this mucosal vaccination might help to protect against TB, and SeV85AB holds notable promise to improve BCG’s protective efficacy in a prime-boost immunization regimen., Hu et al. demonstrate that a Sendai virus-based anti-tuberculosis vaccine, SeV85AB, establishes strong CD8+ resident memory T (TRM) cell immunity in the lung parenchyma when delivered intranasally. This strategy compensates for the weakness of BCG in a prime-boost model and results in markedly enhanced protection against Mycobacterium tuberculosis challenge.

Cytotoxic T lymphocyte (CTL) responses play a central role in viral suppression in human immunodeficiency virus (HIV) infections. Prophylactic vaccination resulting in effective CTL responses after viral exposure would contribute to HIV control. It is important to know how CTL memory induction by vaccination affects postexposure CTL responses. We previously showed vaccine-based control of a simian immunodeficiency virus (SIV) challenge in a group of Burmese rhesus macaques sharing a major histocompatibility complex class I haplotype. Gag 206-216 and Gag 241-249 epitope-specific CTL responses were responsible for this control. In the present study, we show the impact of individual epitope-specific CTL induction by prophylactic vaccination on postexposure CTL responses. In the acute phase after SIV challenge, dominant Gag 206-216 -specific CTL responses with delayed, naive-derived Gag 241-249 -specific CTL induction were observed in Gag 206-216 epitope-vaccinated animals with prophylactic induction of single Gag 206-216 epitope-specific CTL memory, and vice versa in Gag 241-249 epitope-vaccinated animals with single Gag 241-249 epitope-specific CTL induction. Animals with Gag 206-216 -specific CTL induction by vaccination selected for a Gag 206-216 -specific CTL escape mutation by week 5 and showed significantly less decline of plasma viral loads from week 3 to week 5 than in Gag 241-249 epitope-vaccinated animals without escape mutations. Our results present evidence indicating significant influence of prophylactic vaccination on postexposure CTL immunodominance and cooperation of vaccine antigen-specific and non-vaccine antigen-specific CTL responses, which affects virus control. These findings provide great insights into antigen design for CTL-inducing AIDS vaccines.

Combinations of DNA and recombinant-viral-vector based vaccines are promising AIDS vaccine methods because of their potential for inducing cellular immune responses. It was found that Gag-specific cytotoxic lymphocyte (CTL) responses were associated with lowering viremia in an untreated HIV-1 infected cohort. The main objectives of our studies were the construction of DNA and recombinant Sendai virus vector (rSeV) vaccines containing a gag gene from the prevalent Thailand subtype B strain in China and trying to use these vaccines for therapeutic and prophylactic vaccines. The candidate plasmid DNA vaccine pcDNA3.1(+)-gag and recombinant Sendai virus vaccine (rSeV-gag) were constructed separately. It was verified by Western blotting analysis that both DNA and rSeV-gag vaccines expressed the HIV-1 Gag protein correctly and efficiently. Balb/c mice were immunized with these two vaccines in different administration schemes. HIV-1 Gag-specific CTL responses and antibody levels were detected by intracellular cytokine staining assay and enzyme-linked immunosorbant assay (ELISA) respectively. Combined vaccines in a DNA prime/rSeV-gag boost vaccination regimen induced the strongest and most long-lasting Gag-specific CTL and antibody responses. It maintained relatively high levels even 9 weeks post immunization. This data indicated that the prime-boost regimen with DNA and rSeV-gag vaccines may offer promising HIV vaccine regimens.

A current promising AIDS vaccine strategy is to elicit CD8(+) cytotoxic T lymphocyte (CTL) responses that broadly recognize highly-diversified HIVs. In our previous vaccine trial eliciting simian immunodeficiency virus (SIV) mac239 Gag-specific CTL responses, a group of Burmese rhesus macaques possessing a major histocompatibility complex haplotype 90-120-Ia have shown vaccine-based viral control against a homologous SIVmac239 challenge. Vaccine-induced Gag(206-216) epitope-specific CTL responses exerted strong selective pressure on the virus in this control. Here, we have evaluated in vivo efficacy of vaccine-induced Gag(206-216)-specific CTL responses in two 90-120-Ia-positive macaques against challenge with a heterologous SIVsmE543-3 that has the same Gag(206-216) epitope sequence with SIVmac239. Despite efficient Gag(206-216)-specific CTL induction by vaccination, both vaccinees failed to control SIVsmE543-3 replication and neither of them showed mutations within the Gag(206-216) epitope. Further analysis indicated that Gag(206-216)-specific CTLs failed to show responses against SIVsmE543-3 infection due to a change from aspartate to glutamate at Gag residue 205 immediately preceding the amino terminus of Gag(206-216) epitope. Our results suggest that even vaccine-induced CTL efficacy can be abrogated by a single amino acid change in viral epitope flanking region, underlining the influence of viral epitope flanking sequences on CTL-based AIDS vaccine efficacy.

For development of an effective T cell-based AIDS vaccine, it is critical to define the antigens that elicit the most potent responses. Recent studies have suggested that Gag-specific and possibly Vif/Nef-specific CD8 + T cells can be important in control of the AIDS virus. Here, we tested whether induction of these CD8 + T cells by prophylactic vaccination can result in control of simian immunodeficiency virus (SIV) replication in Burmese rhesus macaques sharing the major histocompatibility complex class I (MHC-I) haplotype 90-010-Ie associated with dominant Nef-specific CD8 + T-cell responses. In the first group vaccinated with Gag-expressing vectors ( n = 5 animals), three animals that showed efficient Gag-specific CD8 + T-cell responses in the acute phase postchallenge controlled SIV replication. In the second group vaccinated with Vif- and Nef-expressing vectors ( n = 6 animals), three animals that elicited Vif-specific CD8 + T-cell responses in the acute phase showed SIV control, whereas the remaining three with Nef-specific but not Vif-specific CD8 + T-cell responses failed to control SIV replication. Analysis of 18 animals, consisting of seven unvaccinated noncontrollers and the 11 vaccinees described above, revealed that the sum of Gag- and Vif-specific CD8 + T-cell frequencies in the acute phase was inversely correlated with plasma viral loads in the chronic phase. Our results suggest that replication of the AIDS virus can be controlled by vaccine-induced subdominant Gag/Vif epitope-specific CD8 + T cells, providing a rationale for the induction of Gag- and/or Vif-specific CD8 + T-cell responses by prophylactic AIDS vaccines.

We here report the results of a Phase I/IIa open-label four dose-escalation clinical study assessing the safety, tolerability, and possible therapeutic efficacy of a single intramuscular administration of DVC1-0101, a new gene transfer vector based on a nontransmissible recombinant Sendai virus (rSeV) expressing the human fibroblast growth factor-2 (FGF-2) gene (rSeV/dF-hFGF2), in patients with peripheral arterial disease (PAD). Gene transfer was done in 12 limbs of 12 patients with rest pain, and three of them had ischemic ulcer(s). No cardiovascular or other serious adverse events (SAEs) caused by gene transfer were detected in the patients over a 6-month follow-up. No infectious viral particles, as assessed by hemagglutination activity, were detected in any patient during the study. No representative elevation of proinflammatory cytokines or plasma FGF-2 was seen. Significant and continuous improvements in Rutherford category, absolute claudication distance (ACD), and rest pain were observed (P < 0.05 to 0.01). To the best of our knowledge, this is the first clinical trial of the use of a gene transfer vector based on rSeV. The single intramuscular administration of DVC1-0101 to PAD patients was safe and well tolerated, and resulted in significant improvements of limb function. Larger pivotal studies are warranted as a next step.

Induction of durable cellular immune responses by vaccination is an important strategy for the control of persistent pathogen infection. Viral vectors are promising vaccine tools for eliciting antigen-specific T-cell responses. Repeated vaccination may contribute to durable memory T-cell induction, but anti-vector antibodies could be an obstacle to its efficacy. We previously developed a Sendai virus (SeV) vector vaccine and showed the potential of this vector for efficient T-cell induction in macaques. Here, we examined whether repeated SeV vector vaccination with short intervals can enhance antigen-specific CD8(+) T-cell responses. Four rhesus macaques possessing the MHC-I haplotype 90-120-Ia were immunized three times with intervals of three weeks. For the vaccination, we used replication-defective F-deleted SeV vectors inducing CD8(+) T-cell responses specific for simian immunodeficiency virus Gag(206-216) and Gag(241-249), which are dominant epitopes restricted by 90-120-Ia-derived MHC-I molecules. All four animals showed higher Gag(206-216)-specific and Gag(241-249)-specific CD8(+) T-cell responses after the third vaccination than those after the first vaccination, indicating enhancement of antigen-specific CD8(+) T-cell responses by the second/third SeV vector vaccination even with short intervals. These results suggest that repeated SeV vector vaccination can contribute to induction of efficient and durable T-cell responses.

Viral vectors are promising vaccine tools for eliciting potent cellular immune responses. Pre-existing anti-vector antibodies, however, can be an obstacle to their clinical use in humans. We previously developed a Sendai virus (SeV) vector vaccine and showed the potential of this vector for efficient CD8 + T-cell induction in macaques. Here, we investigated the immunogenicity of SeV vector vaccination in the presence of anti-SeV antibodies. We compared antigen-specific CD8 + T-cell responses after intranasal or intramuscular immunization with a lower dose (one-tenth of that in our previous studies) of SeV vector expressing simian immunodeficiency virus Gag antigen (SeV-Gag) between naive and pre-SeV-infected cynomolgus macaques. Intranasal SeV-Gag immunization efficiently elicited Gag-specific CD8 + T-cell responses not only in naive but also in pre-SeV-infected animals. In contrast, intramuscular SeV-Gag immunization induced Gag-specific CD8 + T-cell responses efficiently in naive but not in pre-SeV-infected animals. These results indicate that both intranasal and intramuscular SeV administrations are equivalently immunogenic in the absence of anti-SeV antibodies, whereas intranasal SeV vaccination is more immunogenic than intramuscular in the presence of anti-SeV antibodies. It is inferred from a recent report investigating the prevalence of anti-SeV antibodies in humans that SeV-specific neutralizing titers in more than 70% of people are no more than those at the SeV-Gag vaccination in pre-SeV-infected macaques in the present study. Taken together, this study implies the potential of intranasal SeV vector vaccination to induce CD8 + T-cell responses even in humans, suggesting a rationale for proceeding to a vaccine clinical trial using this vector.

Cytotoxic T lymphocyte (CTL) responses are crucial for the control of human and simian immunodeficiency virus (HIV and SIV) replication. A promising AIDS vaccine strategy is to induce CTL memory resulting in more effective CTL responses post-viral exposure compared to those in natural HIV infections. We previously developed a CTL-inducing vaccine and showed SIV control in some vaccinated rhesus macaques. These vaccine-based SIV controllers elicited vaccine antigen-specific CTL responses dominantly in the acute phase post-challenge. Here, we examined CTL responses post-challenge in those vaccinated animals that failed to control SIV replication. Unvaccinated rhesus macaques possessing the major histocompatibility complex class I haplotype 90-088-Ij dominantly elicited SIV non-Gag antigen-specific CTL responses after SIV challenge, while those induced with Gag-specific CTL memory by prophylactic vaccination failed to control SIV replication with dominant Gag-specific CTL responses in the acute phase, indicating dominant induction of vaccine antigen-specific CTL responses post-challenge even in non-controllers. Further analysis suggested that prophylactic vaccination results in dominant induction of vaccine antigen-specific CTL responses post-viral exposure but delays SIV non-vaccine antigen-specific CTL responses. These results imply a significant influence of prophylactic vaccination on CTL immunodominance post-viral exposure, providing insights into antigen design in development of a CTL-inducing AIDS vaccine.

Efficient expression of HIV-1 structural gene involves regulation by viral Rev and Rev-responsive elements (RRE). Messenger RNAs of wild-type HIV-1 structural genes are either retained in the nucleus or degraded rapidly in the absence of Rev/RRE; therefore little protein can be expressed. Modifying HIV-1 genes is an excellent approach to circumvent this problem, but it is a laborious and costly process. Using certain vectors to deliver wild-type genes may be a promising approach. In this study, the wild-type and modified gag genes, derived from Chinese HIV-1 isolates, were separately constructed in both plasmid DNA and recombinant Sendai virus vectors (rSeV). The expression and immunogenicity of the wild-type and modified gag genes were compared. The results showed that efficient expression of the modified gag gene could be achieved by transfection with DNA and infection with rSeV, but the efficient expression of wild-type gag could only be achieved by rSeV. In addition, the rSeV expressing wild-type gag elicited similar Gag-specific immune responses with modified gag in both SeV/SeV and DNA-prime/rSeV-boost schemes. However, SeV/SeV failed to produce Gag-specific responses as robust as DNA/rSeV. Then the SeV-specific humoral and cellular immune responses were evaluated just before the second rSeV vaccine immunization. It was found that anti-SeV neutralizing antibody was very low but the SeV-specific cellular response was strong. Efficient expression of wild-type HIV-1 structural genes may make the SeV vector a useful tool for the HIV-1 vaccine research, but the strong SeV-specific cellular immune responses may impair the efficacy of SeV-SeV vaccination scheme.

Viral vectors are promising vaccine tools for eliciting antigen-specific T-cell responses. We previously showed the potential of recombinant Sendai virus (SeV) vectors to induce virus-specific T-cell responses in macaque AIDS models. Here, we have evaluated the immunogenicity of replication-competent V-knocked-out and replication-defective F-deleted SeV vectors in macaques. Intranasal replication-competent and replication-defective SeV immunizations both elicited robust systemic antigen-specific T-cell responses, whereas the responses induced by the former were more durable than those by the latter. However, even the latter-induced T-cell responses remained detectable in a local, retropharyngeal lymph node two months after the immunization. These findings are useful for establishment of a vaccine protocol using SeV vectors.

To study the immune responses elicited by multiple vectors and develop vaccines strategies against prevalent HIV-1 strains in China, we have examined the potency of vaccine regimens of plasmid DNA, adenovirus, and Sendai virus vectors expressing HIV-1 gag consensus sequence of HIV-1 isolates from China for inducing specific immune responses. In BALB/c mice, combination of these vectors induced higher Gag-specific cellular immune response than any regimen using single vector alone. The prime-boost-boost regimen consisting of the triple heterologous vectors induced Gag-specific T-cell responses the most efficiently. In rhesus macaques, the prime-boost-boost regimen induced potent Gag-specific cellular immune responses as well as long lasting humoral immune response, and each booster resulted in rapid and efficient expansion of Gag-specific T cells. These results indicate that this prime-boost-boost regimen using triple heterologous vectors is a promising AIDS vaccine candidate for efficiently inducing HIV-1-specific cellular and humoral immune responses. Its further studies as a promising scheme for therapeutic and/or prophylactic HIV-1 vaccines should be grounded.

Several major histocompatibility complex class I (MHC-I) alleles are associated with lower viral loads and slower disease progression in human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections. Immune-correlates analyses in these MHC-I-related HIV/SIV controllers would lead to elucidation of the mechanism for viral control. Viral control associated with some protective MHC-I alleles is attributed to CD8+ T-cell responses targeting Gag epitopes. We have been trying to know the mechanism of SIV control in multiple groups of Burmese rhesus macaques sharing MHC-I genotypes at the haplotype level. Here, we found a protective MHC-I haplotype, 90-010-Id (D), which is not associated with dominant Gag-specific CD8+ T-cell responses. Viral loads in five D+ animals became significantly lower than those in our previous cohorts after 6 months. Most D+ animals showed predominant Nef-specific but not Gag-specific CD8+ T-cell responses after SIV challenge. Further analyses suggested two Nef-epitope-specific CD8+ T-cell responses exerting strong suppressive pressure on SIV replication. Another set of five D+ animals that received a prophylactic vaccine using a Gag-expressing Sendai virus vector showed significantly reduced viral loads compared to unvaccinated D+ animals at 3 months, suggesting rapid SIV control by Gag-specific CD8+ T-cell responses in addition to Nef-specific ones. These results present a pattern of SIV control with involvement of non-Gag antigen-specific CD8+ T-cell responses.