A novel recombinant adeno-associated virus vaccine induces a long-term humoral immune response to human immunodeficiency virus.

Recombinant adeno-associated virus (AAV) has attracted tremendous interest as a promising vector for gene delivery. In this study we have developed an HIV-1 vaccine, using an AAV vector expressing HIV-1 env, tat, and rev genes (AAV-HIV vector). A single injection of the AAV-HIV vector induced strong production of HIV-1-specific serum IgG and fecal secretory IgA antibodies as well as MHC class I-restricted CTL activity in BALB/c mice. The titer of HIV-1-specific serum IgG remained stable for 10 months. When AAV-HIV vector was coadministered with AAV-IL2 vector, the HIV-specific cell-mediated immunity (CMI) was significantly enhanced. Boosting with AAV-HIV vector strongly enhanced the humoral response. Furthermore, the mouse antisera neutralized an HIV-1 homologous strain, and BALB/c mice immunized via the intranasal route with an AAV vector expressing the influenza virus hemagglutinin (HA) gene showed protective immunity against homologous influenza virus challenge. These results demonstrate that AAV-HIV vector immunization may provide a novel and promising HIV vaccination strategy.

Cocaine abuse and HIV-1 infection: epidemiology and neuropathogenesis.

The epidemiology of cocaine abuse and potential relationships of cocaine withdrawal to human immunodeficiency virus type 1 (HIV-1)-associated dementia (HAD) are discussed. Neuroendocrinological changes in HIV-1 infection of the central nervous system (CNS) are discussed with the relevant impact of cocaine abuse. HIV-1 load in the brain tissue of infected substance users is described along with possible associations with neuropathology and HAD. Finally, the molecular epidemiology and sequence heterogeneity of HIV-1 and their implications for neuropathogenesis are summarized. The complex context of addressing cocaine abuse in the setting of HIV-1 infection appears more tractable when decomposed into its components.

Evidence of HIV type 1 glycoprotein 120 binding to recombinant N-methyl-D-aspartate receptor subunits expressed in a baculovirus system.

Activation of the N-methyl-D-aspartate (NMDA) receptor by HIV-1 envelope glycoprotein 120 (gp120) is thought to represent at least one of the pathways causing neuronal damage in AIDS patients. In the present study, recombinant gp120 binding to NMDA receptor subunits expressed in a baculovirus system was examined by immunocytochemistry and a binding assay, using horseradish peroxidase (HRP)-conjugated and 125I-labeled recombinant gp120, respectively. We found that recombinant gp120 binds to Sf21 cells expressing epsilon1/zeta1 or epsilon2/zeta1 combined NMDA receptor subunits, but not to Sf21 cells infected with mock virus or Sf21 cells expressing a single epsilon1, epsilon2, or zeta1 NMDA receptor subunit. The binding was strongly blocked by unlabeled recombinant gp120, monoclonal anti-HIV-1 gp160 antibody, and a mixture of anti-epsilon1/epsilon2 and anti-zeta1 antibodies. The same results were obtained by flow cytometric analysis. These data suggest that HIV-1 gp120 may directly bind to the NMDA receptor. This evidence enhances our understanding of the mechanism of HIV-1-induced neuronal damage in AIDS patients.

Independent evolution of HIV type 1 in different brain regions.

HIV-1-associated brain pathology exhibits regional variability and we therefore studied the genetic differences in the V1-V5 domains of the HIV env gene in up to four regions of brain (frontal lobe, basal ganglia, medial temporal lobe, and nonmedial temporal lobe) from three patients. We found that in each separate brain region HIV-1 forms different quasispecies and that there is little gene flow among these regions. In further support of brain region-specific evolution of HIV-1, we analyzed amino acid signatures in these clones. In addition to known amino acid signatures associated with macrophage tropism and the lack of syncytium formation, we found 15 majority amino acid signature patterns from the V1-V5 env sequences associated with the neuroanatomical regions analyzed from the three individuals. Furthermore, on average, intrabrain genetic distances for the HIV-1 env were estimated to be much smaller than genetic distances between brain regions. Specific strains of HIV-1 may be neurotropic or neuroinvasive (replication preference in brain tissue) and may contribute to pathology, cognitive loss, and neuropsychiatric disease.

Cationic liposomes are a strong adjuvant for a DNA vaccine of human immunodeficiency virus type 1.

Liposomes have been widely used to enhance the immune response. In the present investigation, we studied their in vivo immunomodulation of an HIV-1-specific DNA vaccine candidate (pCMV160/REV) constructed with the cytomegalovirus (CMV) promoter-conjugated HIV-1 env and rev DNA plasmids. By immunizing with pCMV160/REV and cationic liposomes through various routes (intramuscular, intraperitoneal, subcutaneous, intradermal, and intranasal), we induced higher levels of both antibody production and delayed-type hypersensitivity (DTH) than by using DNA vaccine alone. The HIV-1-specific cytotoxic T lymphocyte (CTL) activity was observed to be stronger on immunization with the DNA vaccine and cationic liposome combination. The intramuscular, intraperitoneal, and intranasal inoculation routes were more effective in inducing strong DTH and antibody responses than the subcutaneous and intradermal routes. Taken together, these results suggest that cationic liposomes can be highly effective when used with DNA vaccines and administered by various routes.

Arginine-481 mutation abolishes ligand-binding of the AMPA-selective glutamate receptor channel alpha1-subunit.

Arginine-481 is located in the putative agonist-binding region preceding the putative transmembrane segment M1 of the alpha1-subunit of the AMPA-selective glutamate receptor (GluR) channel. This amino acid is completely conserved among GluR proteins. A site-directed mutagenesis study using a baculovirus expression system showed that substitution of glutamate, glutamine and lysine for arginine-481 of the recombinant alpha1-subunit protein abolishes binding to [3H]AMPA completely. The present study provides the first direct experimental evidence that the conserved charged arginine-481 residue is essential, directly or indirectly, for the acquisition of ligand-binding activity by the receptor protein.

Erythrocyte delta-aminolevulinic acid dehydratase genotype and other mechanisms affecting workers' susceptibility to lead.

In this study, the role of delta-aminolevulinic acid dehydratase (ALAD) variants in lead susceptibility was examined. The study subjects comprised 223 male workers, and the relationship between their blood lead level and erythrocyte ALAD activity or plasma/urine delta-aminolevulinic acid level was studied. Leukocyte specimens from 11 workers, whose erythrocyte ALAD activities were as low as one-fifth that of the other normal workers, were subjected to analyses of their ALAD and ALAD alleles. Further, the entire exon fragment of the ALAD gene was analyzed by polymerase chain reaction, and the reaction product was used as a target for direct DNA sequencing. Genomic DNA analysis revealed that all 11 workers had the ALAD allele, whereas the entire ALAD gene analysis failed to indicate other variants, except for the Rsa I site. The depletion in erythrocyte ALAD activity was not found to be caused by the ALAD allele.

[Seroepidemiological study of human rotavirus in northern part of Tokyo from 1990 to 1992 by using reverse transcription-polymerase chain reaction].

A seroepidemiological study of rotavirus was conducted in the northern part of Tokyo from 1990 to 1992 by using reverse transcription-polymerase chain reaction (RT-PCR). G1 and G3 types were detected in the winter between 1990 and 1991, however G1 type was appeared mainly in the winter between 1991 and 1992. G3 type was observed as the main type during the winter in the 10 year survey in the Tokyo area for the first time. RT-PCR was useful in the seroepidemiological studies of rotavirus infection.

Prime-boost vaccination with plasmid DNA and a chimeric adenovirus type 5 vector with type 35 fiber induces protective immunity against HIV.

Immunization involving a DNA vaccine prime followed by an adenovirus type 5 (Ad5) boost elicited a protective immune response against SHIV challenge in monkeys. However, the hepatocellular tropism of Ad5 limits the safety of this viral vector. This study examines the safety and immunogenicity of a replication-defective chimeric Ad5 vector with the Ad35 fiber (Ad5/35) in BALB/c mice and rhesus monkeys. This novel Ad5/35 vector showed minimal hepatotoxicity after intramuscular administration with the novel Ad5/35 vector. In addition, an Ad5/35 vector expressing HIV Env gp160 protein (Ad5/35-HIV) generated strong HIV-specific immune responses in both animal models. Priming with a DNA vaccine followed by Ad5/35-HIV boosting yielded protection against a gp160-expressing vaccinia virus challenge in BALB/c mice. The Ad5/35-HIV vector was significantly less susceptible to the pre-existing Ad5 immunity than a comparable Ad5 vector. These findings indicate that an Ad5/35 vector-based HIV vaccine may be of considerable value for clinical use.

Genetic variation in VP7 gene of human rotavirus serotype 1 (G1 type) isolated in Japan and China.

Sequence analysis of the gene encoding the major neutralization glycoprotein (VP7) was performed on 12 human isolates of serotype 1 of rotavirus in Japan and China. They were examined for genetic variations among serotype 1 isolates. Comparative studies of their nucleotide and deduced amino acid sequences between the 12 isolates and the Wa strain revealed an overall homology of more than 92 and 96%, respectively. Higher degrees of homologies were observed between Wa and 2 strains (K1 and K2) in Tokyo, 1979-1980, than between Wa and recent isolated strains in Tokyo and in China. In our isolates, a total of 16 amino acid residues frequently converted to another amino acid. Six amino acid residues belonging to the major neutralizing epitope regions (B, D, and E in this communication) frequently converted. From these data three subtypes (subtypes A, B, and intermediate) were suggested to be divided. Whether these differences are an important mechanism in the epidemiology of rotaviruses requires further investigation.

Detection and sequencing of rotavirus VP7 gene from human materials (stools, sera, cerebrospinal fluids, and throat swabs) by reverse transcription and PCR.

Human rotavirus RNAs from stool samples, sera, cerebrospinal fluids, and throat swabs of 15 children with rotavirus gastroenteritis were detected and serotyped by reverse transcription and PCR. The reverse transcription-PCR method may allow us to consider rotavirus infections in other parts of the body in addition to the gastrointestinal tract. Moreover, sequence analysis of the VP7 gene was performed on seven samples (one stool, two serum, three cerebrospinal fluid, and 1 throat swab sample). There were no appreciable differences in viral sequences between samples from cerebrospinal fluids, sera, or stools.

Immunomodulation by apoptosis-inducing caspases for an influenza DNA vaccine delivered by gene gun.

Apoptosis-inducing caspases have been tested for immunomodulatory effect on a gene gun-delivered DNA vaccine which expresses influenza hemagglutinin. Attenuated murine caspase 2 and a chimera of murine caspase 2 prodomain and human caspase 3 strongly enhanced humoral and cell-mediated immune response to hemagglutinin when they were co-administered with an immunogen DNA. In contrast, wild-type caspases did not enhance the DNA-raised immune response. Caspase dose-dependent antibody response curve revealed that the antibody level was in inverse relation to the amount of administered caspase. These findings indicate that bland apoptosis of antigen-harboring cells can elicit enhanced immune responses. Extensive apoptosis interferes with the generation of immune response. Gene gun delivery involving caspases elicited type-2 immune responses that characterized with dominant IL-4 and IgG1 production. ELISPOT assays showed that CD4 T cells were preferentially activated, while CD8 T cell response remained at marginal level. Using attenuated caspases for gene gun DNA vaccination is a useful approach to amplify type-2 immune responses.

Parvalbumin and calbindin D-28 k immunoreactivity in dorsal root ganglia in acquired immunodeficiency syndrome.

Various degrees of neuronal degeneration have been found in lumbosacral dorsal root ganglia of patients with acquired immunodeficiency syndrome (AIDS). To characterize the subpopulations of primary sensory neurons affected in AIDS, we immunostained dorsal root ganglion tissues from 11 AIDS patients and six controls using antibodies to the calcium binding proteins, parvalbumin and calbindin D-28 k. In controls, the proportion of neurons containing parvalbumin and calbindin was 18.0% and 22.4%, respectively. The majority of parvalbumin-positive neurons, which are thought to be proprioceptive neurons, were of medium to large size, while calbindin was found in both large- and small-sized neurons. The density of parvalbumin-immunoreactive neurons was reduced by 7.3% in AIDS patients, but the density of calbindin-immunoreactive neurons was preserved. Furthermore, in AIDS cases, the number of parvalbumin-positive neurons was reduced more in dorsal root ganglia in which human immunodeficiency virus (HIV) antigen was detected than in HIV-negative ganglia. These results suggest that specific subpopulations of sensory neurons positive for parvalbumin may be differentially affected over the course of AIDS, and that this could be related to peripheral neuropathy which frequently occurs in the late stages of AIDS.

Immunization of RANTES expression plasmid with a DNA vaccine enhances HIV-1-specific immunity.

Cytokines play important roles in regulating immune response. This study evaluated the adjuvant effect of an expression plasmid encoding RANTES (regulated on activation normal T-cell expressed and secreted) chemokine on the immunity induced by a DNA vaccine. This vaccine consists of expression plasmids encoding the env and rev genes of human immunodeficiency virus type 1 (HIV-1). DNA vaccination with RANTES plasmid induced significantly higher titers of serum HIV-1-specific IgG and IgG2a antibodies than DNA vaccination alone on both intramuscular and intranasal immunization. This combination also increased HIV-1-specific cytotoxic T lymphocyte activity and delayed-type hypersensitivity. Intranasal immunization induced a higher titer of fecal secretory IgA antibody than intramuscular immunization. These results demonstrate that coadministration of RANTES plasmid dominantly induced HIV-1-specific cell-mediated immunity.

IL-15 expression plasmid enhances cell-mediated immunity induced by an HIV-1 DNA vaccine.

Cytokines are powerful regulators of the immune response. In this study, an HIV-1 envelope DNA vaccine and interleukin 15 (IL-15) expression plasmid were intranasally administered to mice. A significant increase in the HIV-1-specific DTH response and CTL activity, and decrease in the serum IgG/IgG2a ratio was observed in the group which received DNA vaccine and IL-15 expression plasmid compared to DNA vaccination alone. Restimulated immune lymphoid cells from mice which received both agents showed enhanced production of interferon-gamma (IFN-gamma) and reduced secretion of IL-4. However, administration of DNA vaccine with IL-15 and IL-2 or IL-12 expression plasmids did not alter the effect of IL-15 expression plasmid on the DNA vaccine. These results indicate that intranasal administration of DNA vaccine and IL-15 expression plasmid is capable of enhancing the T helper type 1 (Th1) dependent HIV-1-specific cell-mediated immunity, and that the IL-15 and IL-2 or IL-12 expression plasmids may not have a synergistic effect on the immune response induced by DNA vaccine in vivo.

Immunologic characterization of HIV-specific DNA vaccine.

We developed a method for applying HIV-1 DNA vaccine topically in mice. Topical application of DNA vaccine to the skin is useful against infections. To find a less expensive and less cumbersome vaccination method, we administered HIV-1 DNA vaccine to the skin of mice after elimination of keratinocytes using a fast-acting adhesive. HIV-1 DNA vaccine induced high levels of both humoral and cell-mediated immune activity against HIV-1 envelope antigen. A high level of HIV-1-specific cytotoxic T lymphocyte response was also observed, and a high level of IFN-gamma and IL-4 production was induced by the improved skin application of DNA vaccine. High levels of both HIV-specific cytotoxic T lymphocyte and delayed type hypersensitivity in topical application were induced by coadministration of the DNA vaccine with IL-12 expression plasmids and granulocyte-macrophage colony-stimulating factor expression plasmids. These immune responses were inhibited by intradermal injection of anti-CD11c or anti-I-A/I-E antibody. Therefore, topical administration of DNA vaccine is an effective route, and may be very useful for the prevention of infectious diseases.