Nanogel-based pneumococcal surface protein A nasal vaccine induces microRNA-associated Th17 cell responses with neutralizing antibodies against Streptococcus pneumoniae in macaques.

We previously established a nanosized nasal vaccine delivery system by using a cationic cholesteryl group-bearing pullulan nanogel (cCHP nanogel), which is a universal protein-based antigen-delivery vehicle for adjuvant-free nasal vaccination. In the present study, we examined the central nervous system safety and efficacy of nasal vaccination with our developed cCHP nanogel containing pneumococcal surface protein A (PspA-nanogel) against pneumococcal infection in nonhuman primates. When [(18)F]-labeled PspA-nanogel was nasally administered to a rhesus macaque (Macaca mulatta), longer-term retention of PspA was noted in the nasal cavity when compared with administration of PspA alone. Of importance, no deposition of [(18)F]-PspA was seen in the olfactory bulbs or brain. Nasal PspA-nanogel vaccination effectively induced PspA-specific serum IgG with protective activity and mucosal secretory IgA (SIgA) Ab responses in cynomolgus macaques (Macaca fascicularis). Nasal PspA-nanogel-induced immune responses were mediated through T-helper (Th) 2 and Th17 cytokine responses concomitantly with marked increases in the levels of miR-181a and miR-326 in the serum and respiratory tract tissues, respectively, of the macaques. These results demonstrate that nasal PspA-nanogel vaccination is a safe and effective strategy for the development of a nasal vaccine for the prevention of pneumonia in humans.

Periventricular leucomalacia (PVL)-like lesions in two neonatal cynomolgus monkeys (Macaca fascicularis).

Periventricular leucomalacia (PVL) is a lesion of immature cerebral white matter that occurs in the perinatal period. In man, PVL is the predominant form of brain injury and a cause of cerebral palsy and cognitive deficits in premature infants. PVL affects fetuses and newborns, particularly those who have undergone oxygen deprivation as may occur in premature birth. Many clinical and pathological studies of PVL have been performed in man, but there is no clear definition of PVL in animals. A few spontaneous PVL-like cases in puppies or experimental cases in other animal species have been reported. The present study reports the histopathological and immunohistochemical features of PVL-like lesions in two neonatal cynomolgus monkeys. In both cases, there was cerebral white matter necrosis with marked infiltration of lipid-laden phagocytes and a reduction of neurons in the cerebral cortex. In case 1 there was extensive cavitation of the cerebral white matter. In case 2 there was reactive astrocytosis associated with a decrease in oligodendroglial cells and a decrease in cerebral white matter myelin. To our knowledge, this is the first report of PVL-like leucoencephalomalacia in non-human primates.

IL-4/IL-13 antagonist DNA vaccination successfully suppresses Th2 type chronic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is a chronic disease with a Th2-type-cytokine dominant profile. Several cytokines and related peptides have been used for the treatment of AD but they were ineffective because of their limited biological half-life. We have recently developed a highly efficient mouse dominant negative interleukin (IL)-4/IL-13 antagonist (IL-4DM), which blocks both IL-4 and IL-13 signal transductions. OBJECTIVE: To examine the effects of IL-4DM in vivo in an AD model induced by the repeated exhibition of oxazolone (OX). METHODS: Plasmid DNA was injected intraperitoneally to cause an experimental AD-like dermatitis. The effect was evaluated by ear thickness, histological findings, and mast cells counts in the inflamed skin. The plasma IgE and histamine levels were measured. Cytokine production in skin and splenocytes were also analysed. RESULTS: Mice treated with control plasmid developed marked dermatitis with mast cells and eosinophil infiltration, and had increased plasma IgE and histamine levels with a Th2 type splenocyte cytokine profile. Treatment with mouse IL-4 DNA augmented the ear swelling and thickness with an increased dermal eosinophil count, plasma histamine level, and production of splenocyte IL-4. However, IL-4DM treatment successfully controlled the dermatitis, decreased the mast cell and eosinophil count, and suppressed plasma IgE and histamine levels. Splenocytes produced an increased level of IFN-gamma. CONCLUSION: These data showed that the simultaneous suppression of IL-4/IL-13 signals successfully controlled Th2-type chronic dermatitis. IL-4DM DNA treatment is a potent therapy for AD and related diseases.

Acute megakaryocytic leukaemia (AMKL)-like disease in a cynomolgus monkey (Macaca fascicularis).

A 5-year-old male cynomolgus monkey (Macaca fascicularis) with a clinical history of bleeding tendency, severe anaemia, thrombocytopenia and elevated serum concentration of liver-related enzymes was examined post mortem. Ecchymotic haemorrhages were present on the left eyelid and forehead. The liver, kidney and spleen were markedly enlarged and the kidneys had capsular petechiae. Microscopically, numerous atypical cells resembling myeloid cells were observed in the bone marrow, and myelofibrosis was present. Atypical cells were also present in the blood vessels of the liver, kidney, spleen, lymph nodes, lung, heart, bladder, adrenal gland and brain. Some neoplastic cells had oval or pleomorphic macronuclei and others were multinucleated. Immunohistochemically, the majority of the neoplastic cells had granular cytoplasmic expression of the megakaryocyte-associated antigens Von Willebrand Factor and CD61-IIIa, but were negative for myeloperoxidase. A diagnosis of acute megakaryocytic leukaemia (AMKL)-like disease was made. This would appear to be the first report of AMKL-like disease in non-human primates. This monkey was infected with simian retrovirus type D and it is possible that this viral infection was associated with the development of neoplasia.

Congenital cystic adenomatoid-like malformation in a cynomolgus monkey (Macaca fascicularis).

Congenital cystic adenomatoid malformation (CCAM) is a developmental lung abnormality characterized by abnormal proliferation of mesenchymal elements and failure of bronchiolar structures to mature, ultimately resulting in the compression of normal pulmonary tissue and mediastinal shift with rapid expansion of cysts. Although various clinical and pathologic studies of CCAM in humans exist, CCAM has yet to be reported in animals, even in nonhuman primates. In the present study, histopathologic analyses of a neonatal cynomolgus monkey that died 17 days after birth revealed that normal lung architecture was replaced by disorganized overgrowths of cysts lined with simple cuboidal epithelium. The epithelium projected a few ciliates into the air spaces and produced mucus. To our knowledge, this is the first case study describing CCAM or a CCAM-like lesion in nonhuman primates.

DNA vaccine-encapsulated virus-like particles derived from an orally transmissible virus stimulate mucosal and systemic immune responses by oral administration.

Delivery of foreign genes to the digestive tract mucosa by oral administration of nonreplicating gene transfer vectors would be a very useful method for vaccination and gene therapy. However, there have been few reports on suitable vectors. In the present study, we found that plasmid DNA can be packaged in vitro into a virus-like particle (VLP) composed of open reading frame 2 of hepatitis E virus, which is an orally transmissible virus, and that these VLPs can deliver this foreign DNA to the intestinal mucosa in vivo. The delivery of plasmid DNA to the mucosa of the small intestine was confirmed by the results of immunohistochemical analyses using an expression plasmid encoding human immunodeficiency virus env (HIV env) gp120. After oral administration of VLPs loaded with HIV env cDNA, significant levels of specific IgG and IgA to HIV env in fecal extracts and sera were found. Moreover, mice used in this study exhibited cytotoxic T-lymphocyte responses specific to HIV env in the spleen, Payer's patches and mesenteric lymph nodes. These findings suggest that VLPs derived from orally transmissible viruses can be used as vectors for delivery of genes to mucosal tissue by oral administration for the purpose of DNA vaccination and gene therapy.

A single administration of interleukin-4 antagonistic mutant DNA inhibits allergic airway inflammation in a mouse model of asthma.

Interleukin 4 (IL-4) is essential for the switching of B cells to IgE antibody production and for the maturation of T helper (Th) cells toward the Th2 phenotype. These mechanisms are thought to play a crucial role in the pathogenesis of the allergic airway inflammation observed in asthma. In the present study, we examined the anti-inflammatory effects of DNA administration of murine IL-4 mutant Q116D/Y119D (IL-4 double mutant, IL-4DM), which binds to the IL-4 receptor alpha and is an antagonist for IL-4. Immunization of BALB/c mice with alum-adsorbed ovalbumin (OVA) followed by aspiration with aerosolized OVA resulted in the development of allergic airway inflammation. A single administration of IL-4DM DNA before the aerosolized OVA challenge protected the mice from the subsequent induction of allergic airway inflammation. Serum IgE level and extent of eosinophil infiltration in bronchoalveolar lavage (BAL) from IL-4DM DNA-administered mice were significantly lower than those in BAL from control plasmid-immunized mice. In our study, IL-4 or IL-4 mutants were not detected in sera from mice that had received a single administration of IL-4DM DNA. The results of this study provide evidence for the potential utility of IL-4 mutant antagonist DNA inoculation as an approach to gene therapy for asthma.

Interleukin-4-expressing plasmid DNA inhibits reovirus type-2-triggered autoimmune insulitis in DBA/1 J suckling mice.

In this study we have examined the effect of systemic administration of T helper (Th) 2 cytokines on reovirus type-2 (Reo-2)-triggered Th1-mediated autoimmune insulitis with impaired glucose tolerance (IGT) in DBA/1J suckling mice. We have demonstrated clearly that the systemic administration of both interleukin (IL)-4-expressing plasmid DNA (pIL-4) and recombinant IL-4 (rIL-4) inhibited the development of insulitis with IGT in a dose dependent manner as compared to untreated groups in Reo-2-infected DBA/1J suckling mice. The inhibitory effects of IL-4 on the development of insulitis with IGT and the advantages of pIL-4 as compared to rIL-4 in this model are discussed.

HER2 peptide-specific CD8(+) T cells are proportionally detectable long after multiple DNA vaccinations.

We prepared a plasmid encoding 147 amino acid residues from the N terminus of c-erbB-2/HER2/neu (HER2), which included both a cytotoxic T lymphocyte (CTL) epitope (HER2p63) and a helper epitope (HER2p1), using the mammalian expression vector pCAGGS-New (pCAGGS147HER2). In a parallel analysis with a Tetramer assay and CTL assay, good specificity and sensitivity of a quantitative enzyme-linked immunospot (ELISPOT) assay to detect functional HER2p63-specific CD8(+) T cells were demonstrated after intramuscular immunization of pCAGGS147HER2. In an ELISPOT assay for HER2p63, spots of IFN gamma-producing cells were first detected 10 days after the first immunization, and additional immunizations increased the number of spots. HER2p63-specific CD8(+) T cells were detected over a period of more than 10 months after the last immunization. In hosts receiving more than three immunizations, surprisingly high numbers of specific CD8(+) T cells were persistently detectable. HER2 protein-specific antibodies of IgG class with dominance of IgG2a remain detectable 6 months after single or multiple immunizations. The antibodies however, were not reactive with cell surface HER2 antigens. Total suppression of tumor growth was observed when syngeneic HER2(+) tumor cells (2 x 10(6)) were injected subcutaneously 14 days after a single immunization with pCAGGS147HER2. Furthermore, the number of pulmonary metastases decreased significantly when DNA vaccination was initiated on the day of, or 3 days after, intravenous injection (1 x 10(6) cells).

Induction of effective antitumor immune responses in a mouse bladder tumor model by using DNA of an alpha antigen from mycobacteria.

One of the main objectives of cancer immunotherapy is the activation and increase in number of antitumor effector cells. Recently, genetically modified tumor cell vaccines have been proposed for elicitation of antitumor effector cells. Native alpha antigen (alpha Ag) (also known as MPT59 and antigen 85B) of mycobacteria, which cross-reacts among mycobacteria species, may play an important biological role in host-pathogen interaction because it elicits various helper T-cell type 1 immune responses. To assess the induction of antitumor immune responses by alpha Ag, mouse tumor cell lines transfected with cDNA of alpha Ag from Mycobacterium kansasii were established, and the possibility of producing a tumor cell vaccine for induction of antitumor effects was explored. Transfection of tumor cell lines with an alpha Ag gene lead to primary tumor rejection and the establishment of protective immunity to nontransfected original tumor cell lines in Mycobacterium bovis bacillus Calmette-Gurin (BCG)-primed and unprimed mice. Mice immunized with tumor cell lines transfected with the alpha Ag gene showed delayed-type hypersensitivity responses in vivo and proliferative responses together with induction of interferon-gamma of spleen cells against nontransfected wild-type tumor cell lines in in vitro experiments. Moreover, immunization of mice with alpha Ag-expressing tumor cells elicited tumor-specific and cytotoxic T lymphocyte (CTL) epitope peptide-specific CD8+ CTLs. The results of this study provided evidence of the potential usefulness of alpha Ag in tumor cell vaccines.

Quintuple deglycosylation mutant of simian immunodeficiency virus SIVmac239 in rhesus macaques: robust primary replication, tightly contained chronic infection, and elicitation of potent immunity against the parental wild-type strain.

We previously generated a mutant of simian immunodeficiency virus (SIV) lacking 5 of a total of 22 N-glycans in its external envelope protein gp120 with no impairment in viral replication capability and infectivity in tissue culture cells. Here, we infected rhesus macaques with this mutant and found that it also replicated robustly in the acute phase but was tightly, though not completely, contained in the chronic phase. Thus, a critical requirement for the N-glycans for the full extent of chronic infection was demonstrated. No evidence indicating reversion to a wild type was obtained during the observation period of more than 40 weeks. Monkeys infected with the mutant were found to tolerate a challenge infection with wild-type SIV very well. Analyses of host responses following challenge revealed no neutralizing antibodies against the challenge virus but strong secondary responses of cytotoxic T lymphocytes against multiple antigens, including Gag-Pol, Nef, and Env. Thus, the quintuple deglycosylation mutant appeared to represent a novel class of SIV live attenuated vaccine.

Induction of virus-specific cytotoxic T lymphocytes by in vivo electric administration of peptides.

Generally, major histocompatibility complex (MHC) class I presentation of peptide antigens only occur for proteins' which are actively synthesized and processed intracellularly, so that immunization with a cytotoxic T lymphocyte (CTL) target peptide does not usually elicit effective CTL responses. In the present study, we explored the use of epitope peptides by in vivo electroporation to introduce directly into the cytoplasm for the vaccine elicitation of virus-specific CTLs in a mouse system. BALB/c mice were immunized with human immunodeficiency virus (HIV) env (P18, residues 311-320) or hepatitis C virus (HCV) NS5 (P17, residues 2423-2434) with or without electric pulses. Effector cells against peptide-labeled target cells were elicited in mice immunized with peptides with electric administration but not without electric administration. Moreover, cytolytic activities of CTL against peptide-labeled target cells were enhanced by the addition of plasmid having the immunostimulatory sequence (ISS) or cDNA of the B7-1 molecule in electric administration of peptides. The results of the present study suggest that a peptide vaccine against a virus using electric administration is effective in eliciting virus specific CTLs.

Role of natural killer cells in resistance against friend retrovirus-induced leukemia.

We have previously shown that immunization with a synthetic peptide that contains a single CD4(+) T-cell epitope protects mice against immunosuppressive Friend retrovirus infection. Cells producing infectious Friend virus were rapidly eliminated from the spleens of mice that had been immunized with the single-epitope peptide. However, actual effector mechanisms induced through T-helper-cell responses after Friend virus inoculation were unknown. When cytotoxic effector cells detected in the early phase of Friend retrovirus infection were separated based on their expression of cell surface markers, those lacking CD4 and CD8 but expressing natural killer cell markers were found to constitute the majority of effector cells that lysed Friend virus-induced leukemia cells. Depletion of natural killer cells by injecting anti-asialo-ganglio-N-tetraosylceramide antibody did not affect the number of CD4(+) or CD8(+) T cells in the spleen, virus antigen-specific proliferative responses of CD4(+) T cells, or cytotoxic activity against Friend virus-induced leukemia cells exerted by CD8(+) effector cells. However, the same treatment markedly reduced the killing activity of CD4(-) CD8(-) effector cells and completely abolished the effect of peptide immunization. Although the above enhancement of natural killer cell activity in the early stage of Friend virus infection was also observed in mice given no peptide, these results have demonstrated the importance and requirement of natural killer cells in vaccine-induced resistance against the retroviral infection.

Induction of hepatitis C virus-specific cytotoxic T lymphocytes in mice by an intrahepatic inoculation with an expression plasmid.

We assessed the possibility of intrahepatic inoculation with a plasmid encoding hepatitis C virus (HCV) proteins to elicit HCV-specific cytotoxic T lymphocytes (CTL) in mice as a conventional animal model of HCV infection. BALB/c mice were intrahepatically or intramuscularly inoculated with an expression plasmid DNA encoding HCV structural proteins under the control of the elongation factor 1-alpha promoter. Expressions of HCV-core protein and envelope proteins (E1 and E2) in hepatocytes were detected immunohistochemically 6 days after inoculation. CTL responses were examined using target cells either pulsed with a specific peptide or infected with a recombinant vaccinia virus expressing HCV structural protein. Both intrahepatically and intramuscularly DNA-inoculated mice developed CD8(+), MHC class I-restricted CTL responses that recognized the peptide pulsed as well as HCV proteins expressing target cells. These studies demonstrated the usefulness of a murine model of HCV infection induced by direct intrahepatic DNA inoculation for understanding the immunopathogenic mechanisms in HCV infection.

Suppression of acute viremia by short-term postexposure prophylaxis of simian/human immunodeficiency virus SHIV-RT-infected monkeys with a novel reverse transcriptase inhibitor (GW420867) allows for development of potent antiviral immune responses resulting in efficient containment of infection.

A nonnucleoside reverse transcriptase (RT) inhibitor, GW420867, was tested for postexposure prophylaxis (PEP) in rhesus macaques experimentally infected with 100 50% tissue culture infective doses of a chimeric simian/human immunodeficiency virus (SHIV) containing the RT gene of HIV-1 (SHIV-RT). Animals were either mock treated, or treated for 4 weeks starting at 8 or 24 h postinfection (p.i.) with GW420867. While such therapy led to undetectable plasma viremia in three of six monkeys, a transient plasma viremia was noted in the other three treated animals at 2 to 4 weeks following cessation of therapy. Following this transient viremia all drug-treated animals showed low or undetectable levels of plasma viremia up to the last sample examined at 90 weeks p.i. Despite low and/or undetectable viremia, virus-specific cytotoxic T lymphocyte and viral Env-specific proliferative responses were seen in the peripheral blood mononuclear cells of both mock- and drug-treated animals as early as 3 weeks p.i. Such virus-specific cellular responses, however, were better maintained in the drug-treated animals than the mock-treated animals. In contrast to the virus-specific cellular response, the magnitude and kinetics of virus specific humoral responses appeared to correlate with the detection of viremia. These data support the view that a short-term PEP with GW420867 permits the generation and maintenance of long-lasting virus-specific cell-mediated immune responses while markedly reducing viral loads to undetectable levels for a prolonged period of time (90 weeks) and leads to long-term disease protection. This model provides a unique means to define mechanisms and correlates of disease protection.

Evaluation of a lipopeptide immunogen as a therapeutic in HIV type 1-seropositive individuals.

A 32-amino acid HIV-1 Gag immunogen was assessed for its ability to augment existing virus-specific CTL responses in chronically HIV-1-infected individuals. The immunogen was an HIV-1 synthetic lipopeptide conjugate composed of an N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R)-propyl-N-(R)-cysteinyl] group covalently coupled to a synthetic 32-amino acid Gag peptide containing at least 5 CTL epitopes known to be restricted by HLA-A33, -B8, -B27, -B35, and -Bw62. This potential immunotherapeutic was first determined to be safe in six HIV-1-seropositive subjects, with no adverse clinical effects noted during a 182-day period after administration of a dose of 350 microg. The immunogenicity of this lipopeptide conjugate was then assessed in a pilot study in nine HIV-1-seropositive volunteers with peripheral blood CD4+ lymphocyte counts of >500/microl. Three groups of individuals were studied: HLA-selected subjects who received 350 microg of the immunogen on days 0, 28, and 56 (four subjects); HLA-selected subjects who received a placebo according to a similar inoculation schedule (three subjects); and HLA-mismatched subjects who received the experimental immunogen (two subjects). All subjects were monitored for 26 weeks. After treatment, PBLs from two of the four HLA-selected subjects who received the experimental immunogen showed a transient increase in Gag peptide-specific bulk CTL activity. None of the placebo-vaccinated or vaccinated HLA-mismatched subjects showed any change in bulk Gag peptide-specific CTL activity. However, no consistent decrease in plasma HIV-1 RNA levels was noted in any of the subjects. The present study illustrates that this peptide formulation may not be a sufficiently potent immunogen to significantly augment HIV-1-specific CTLs and to decrease virus load in HIV-1-seropositive individuals.

Malaria infection induces rapid elevation of the soluble Fas ligand level in serum and subsequent T lymphocytopenia: possible factors responsible for the differences in susceptibility of two species of Macaca monkeys to Plasmodium coatneyi infection.

The intraerythrocytic stage of the simian malaria parasite Plasmodium coatneyi (CDC strain) was intravenously inoculated into two species of macaques with different susceptibilities to infection with this parasite, including four Japanese macaques (Macaca fuscata) and three cynomolgus macaques (M. fascicularis). The Japanese macaques infected with P. coatneyi developed severe clinical manifestations similar to those of severe human malaria and eventually became moribund, while the infected cynomolgus macaques, natural hosts of the parasite, exhibited no severe manifestation of disease except anemia and finally recovered from the infection. In the infected Japanese macaques, peripheral CD4(+) and CD8(+) T-cell populations were markedly decreased and fragmentation of chromosomal DNA in peripheral blood mononuclear cells was detected during the terminal period of infection, suggesting that apoptotic cell death was responsible at least in part for the T lymphocytopenia. Furthermore, soluble Fas ligand levels in sera of the infected Japanese macaques increased gradually to a markedly high level of 28. 83 +/- 10.56 pg/ml (n = 4) when the animals became moribund. On the other hand, none of the infected cynomolgus monkeys exhibited either T lymphocytopenia or elevated soluble Fas ligand level. These findings suggest that differences in immune response between the two species of macaque tested accounted for the contrasting outcomes after infection with the same isolate of malarial parasite, and in particular that a profound T lymphocytopenia due to Fas-derived apoptosis played a role in the fatal course of malaria in the infected Japanese macaques.

A single immunization with a plasmid encoding hepatitis C virus (HCV) structural proteins under the elongation factor 1-alpha promoter elicits HCV-specific cytotoxic T-lymphocytes (CTL).

Recent studies have raised the possibility that DNA-based vaccination may prove useful for generating virus-specific cytotoxic T-lymphocytes (CTL) responses. Recently, a plasmid containing the human elongation factor 1alpha(EF1-alpha) promoter, pEF321, was reported to be a versatile expression vector for gene expression in mammalian cells in vitro. In the present study, we assessed the capability of a novel plasmid, pEFCE1E2, encoding hepatitis C virus (HCV) structural proteins (core, E1 and E2) under the EF1-alpha promoter to generate CTL against HCV in vivo. BALB/c mice were immunized with the pEFCE1E2 but not with a plasmid possessing the same cDNA under the cytomegalovirus developed HCV-specific effector cells by a single immunization. These effector cells elicited by pEFCE1E2 immunization were CD8(+) and major histocompatibility complex class I restricted. These studies provided evidence for the potential utility of the EF1-alpha promoter for development of DNA vaccines against HCV infections.

Helper T cell determinant peptide contributes to induction of cellular immune responses by peptide vaccines against hepatitis C virus.

The capacity of novel subunit vaccines to generate cytotoxic T lymphocytes (CTLs) against hepatitis C virus (HCV) was assessed. BALB/c mice were immunized with peptides based on the CTL and helper T cell (Th) epitopes of the HCV core, with a mixture of CTL and Th peptides (CTL+Th) or with a conjugated Th-CTL peptide. Mice immunized with CTL, CTL+Th and Th-CTL peptides, but not those immunized with Th peptide, developed HCV core CTL epitope-specific effector cells. Cytotoxic activity induced by immunization with Th-CTL was much higher than that induced by immunization with CTL+Th or CTL alone. However, rapid and high cytotoxic activities against HCV core were not only detected after immunization with peptides containing the CTL epitope but also as a result of infection with recombinant vaccinia virus carrying the HCV core gene after immunization with the Th epitope alone. Immunization with peptides containing the Th epitope also elicited spleen cell proliferation. This study demonstrates the capacity of both Th and CTL activated peptide vaccines to elicit CD8+, MHC class I-restricted CTLs. The capacity of such CTLs to contribute towards a protective and/or pathogenic immune response against HCV can now be assessed in mouse models.