Ryozaburo Mukai

Infection of macaque monkeys with a chimeric human and simian immunodeficiency virus

Two macaque monkeys were inoculated with a chimeric human and simian immunodeficiency virus carrying the tat, rev, vpu and env genes of human immunodeficiency virus type 1. Infectious virus was recovered from one of the monkeys at 2 and 6 weeks post-infection. The hybrid nature of the isolated viruses was verified by Southern and Western blotting analyses. Both of the monkeys infected with the chimera elicited a humoral antibody response against the virus.

Protection of monkeys vaccinated with vpr- and/or nef-defective simian immunodeficiency virus strain mac/human immunodeficiency virus type 1 chimeric viruses: a potential candidate live-attenuated human AIDS vaccine.

Two simian immunodeficiency virus strain mac (SIVmac)/human immunodeficiency virus type 1(HIV-1) chimeric viruses (SHIVs), designated NM-3 and NM-3n, with env derived from HIV-1 and defective vpr (plus defective nef for NM-3), were inoculated into seven macaques. These macaques were transiently or persistently infected and most of them produced long-lasting neutralizing antibodies and Env-specific killer T cells to HIV-1 with no AIDS-like symptoms. When they were challenged with another SHIV with intact vpr and nef (designated NM-3rN), all were protected as judged by virus recovery, DNA detection by PCR and antibody responses. Anti-HIV-1 Env-specific killer T cells were considered to have played a major role in this protection, but a non-specific defence mechanism as well as specific immunity also appeared to be involved. Thus, these two non-pathogenic SHIVs induced long-lasting protective immunities in macaques, suggesting the possibility of gene-defective SHIVs as attenuated live vaccines for human use.

Transient Expression of CD20 Antigen (Pan B Cell Marker) in Activated Lymph Node T Cells

In contrast to the case of peripheral T cells, the surface expression of CD20 antigen and the expression of CD20 mRNA in monkey lymph node (LN) T cells underwent a noticeable increase when they were cultured with mitogen and interleukin‐2 (IL‐2). To confirm in vivo regulation of CD20 expression during the activation of LN T cells, we examined LNs derived from monkeys experimentally inoculated with simian immunodeficiency virus (SIV). Significant expression of CD20 antigen was detected in the T cells of the LNs at the stage of lymphadenopathy. These findings suggest that lymphocyte activation in the LNs induced expression of the CD20 molecule in some T cells.

Immunogenicity of a Japanese encephalitis DNA vaccine candidate in cynomolgus monkeys

A Japanese encephalitis (JE) vaccine candidate encoding JE virus premembrane (prM) and envelope (E) genes, designated pNJEME, was evaluated for safety and immunogenicity in non-human primate, cynomolgus monkeys. pNJEME was constructed using a vector (pNGVL4a) designed to address some of the safety concerns of DNA vaccine. In two different experiments, two immunizations with 300 microg of pNJEME by intramuscular (i.m.) injection, and 3 microg of pNJEME using a gene gun, and three immunizations by i.m. injection with 500 microg of pNJEME were performed. All the three protocols induced low to high levels of neutralizing antibody, indicating an ability of pNJEME to induce neutralizing antibody in monkeys with a wide individual variation in response to pNJEME. In one experiment designed to compare the DNA vaccine with a commercial inactivated JE vaccine, three immunizations by i.m. inoculation with 300 microg of pNJEME or by gene gun administration with 3 microg of pNJEME induced similar levels of neutralizing antibody to those induced by three immunizations with a human dose of the inactivated vaccine in most monkeys. After intranasal challenge with the Beijing P3 or JaTH160 strain of JE virus, pNJEME-immunized monkeys showed anamnestic neutralizing antibody responses, indicating that pNJEME induced memory B cells which were responsive to infection with JE virus. No systemic and local reactions were observed in any monkeys after i.m. or gene gun inoculations with plasmid DNAs.

Genomic and biological alteration of a human immunodeficiency virus type 1 (HIV-1)-simian immunodeficiency virus strain mac chimera, with HIV-1 Env, recovered from a long-term carrier monkey

A macaque monkey infected with NM-3, a human immunodeficiency virus type 1 (HIV-1)-simian immunodeficiency virus strain mac (SIVmac) chimeric virus with env, rev, tat and vpu derived from HIV-1 and LTR, gag, pol, vif and vpx derived from SIVmac, became a long-term carrier (more than 2.8 years). This monkey produced neutralizing antibodies to the original NM-3 as well as to the parental HIV-1. The virus recovered at 116 weeks replicated more rapidly and productively in macaque peripheral blood mononuclear cells than the original virus. The recovered virus was not neutralized either by antibodies raised early in the monkey or by a neutralizing monoclonal antibody that recognizes the V3 loop of HIV-1 Env, whereas both the early antibodies and the monoclonal antibody neutralized the original NM-3. Analysis of the virus genomic population revealed a few common mutations in the V3 region that caused amino acid changes. These data are consistent with the hypothesis that the virus escaped from the early antibodies and that the observed mutations contributed to this, as with HIV-1-infected humans. The observed mutations could equally well be the result of adaptation to simian cells. These results suggest that the HIV-1-SIVmac chimeric virus will be useful for investigating genetic variation of HIV-1 env and alteration of biological properties in vivo in relation to the host immune response.

CD3 polymorphism in cynomolgus monkeys (Macaca fascicularis)

Abstract: Cynomolgus monkeys were divided into two groups in terms of the reactivity of their lymphocytes with the FN18 monoclonal antibody, which is directed to the CD3 of rhesus monkeys. It was shown that 24 (12.2%) out of 196 monkeys did not have lymphocytes that reacted with the FN18, although T cells from those animals responded well to mitogenic stimulation. We have determined the nucleotide sequences of the CD3δ, CD3γ, and CD3ε chains and found that two amino acids of the CD3ε chain of the FN18 non‐reactive monkeys were different when compared with the FN18 reactive monkeys. Our results indicated that the CD3ε molecule of cynomolgus monkeys is polymorphic at the epitope level, which is recognized by the FN18 monoclonal antibody.

Detection of B Virus Antibody in Monkey Sera Using Glycoprotein D Expressed in Mammalian Cells

ABSTRACT The gene encoding glycoprotein D (gD) of the monkey B virus (Cercopithecine herpesvirus1) was cloned into a mammalian expression vector, pcDNA3.1(−), and the recombinant plasmid DNA was transfected into COS7 cells. The expression of gD in transfected COS7 cells was detected by indirect immunofluorescence assay or radioimmunoprecipitation analysis (RIPA). Although the expressed gD protein was revealed to react well with sera from monkeys naturally infected with B virus by RIPA, some sera showed reduced reactivity when analyzed by the Western blotting (WB) method. Some sera also showed relatively high background when the WB was performed using gD expressed from recombinant plasmid. The mutant gD protein lacking the transmembrane domain (TM) and cytoplasmic tail (CT) was next expressed in COS7 cells. The mutant protein was secreted into culture medium without apparent loss of the antigenicity. Using the secretory form of the gD protein as antigen in dot blot analysis, sera from B virus-infected monkeys were shown to react with the mutant protein without nonspecific reaction. Since the recombinant gD or its derivative lacking TM and CT could be expressed in mammalian cells with proper antigenicity, these antigens appeared to be useful for serological detection of B virus infection in monkeys.

Sequence and functional properties of African green monkey CD4 silencer.

We reported previously that in African green monkey (AGM) CD4 lymphocytes, CD4 mRNA expression undergoes a decrease following in vitro activation, and CD4 cells are therefore subject to loss of CD4 expression on the cell surface. To examine the transcriptional regulation of the CD4 gene in this species. we analyzed the CD4 silencer, which has been identified as a regulatory element responsible for the down regulation of CD4 transcription in CD8 cell lineage cells. Sequence analysis indicated that the CD4 silencer of the AGM was highly homologous to that of humans. However, two nucleotide substitutions were present in one of the nuclear protein binding sites, which was characterized as the FP II site having a strong enhancing effect on transgene expression in CD4 cells. By performing transient transfection assays. we found that the enhancing activities of the CD4 silencer or FP II-containing fragment of the AGM were greatly reduced in a human CD4 cell line as compared to those of human materials. The CD4 mRNA level was significantly decreased in the human CD4 cell line when synthetic oligonucleotide corresponding to the human FP II sequence was added to the culture. These observations imply that FP II-protein interaction might be required for the maintenance of sufficient expression of the CD4 gene, and the enhancing activity mediated by the above interaction might be decreased in the AGM CD4 silencer, due probably to the nucleotide changes occurring at the FP II site.

Identification of an amino acid responsible for the CD3 polymorphism in cynomolgus monkeys (Macaca fascicularis).

Abstract: The FN18 monoclonal antibody (mAb), directed to CD3 molecules, did not react with the lymphocytes of some cynomolgus monkeys (Macaca fascicularis), because of the polymorphism of the CD3ɛ chain. The epitope recognized by the FN18 mAb was successfully expressed on COS7 cells upon transfection of plasmid DNA coding for the CD3ɛ derived from T cells of a FN18 positive cynomolgus monkey. By construction and expression of plasmid DNA encoding the mutant CD3ɛ, the amino acid residue at position 67 was demonstrated to be involved in the formation of an epitope recognizable by the FN18 mAb.

Detection of CD3ɛ polymorphism in cynomolgus monkeys by a method based on RFLP

Abstract:  We previously reported that peripheral lymphocytes from about 12% of cynomolgus monkeys lacked reactivity with anti‐rhesus monkey CD3 monoclonal antibody (FN18). The nucleotide sequence analysis of the genes encoding CD3 component proteins revealed that a single amino acid substitutions found in the CD3ɛ chain determined the phenotype. In this study, we attempted to develop a method based on the restriction fragment length polymorphism (RFLP) and apply it for determination of the genotypes of individual monkeys. Comparison of the phenotype determined by fluorescence‐activated cell sorter analysis with the genotype determined by RFLP analysis revealed that the FN18 ‐positive trait was dominant over the FN18‐negative trait. It was also revealed that allele frequency was significantly different among macaques depending on the geographical region where their ancestors were derived from.