Rapid quantitative detection system for measles virus-neutralizing antibodies using HiBiT-tagged virus-like particles.

Immunological testing to detect neutralizing antibodies (NAbs) is important in measles (MV) infection control. Currently, the plaque reduction neutralization test is the only credible method for measuring actual virus NAbs; however, its feasibility is hampered by drawbacks, such as long turnaround times, low throughput, and the need for laboratory biosafety equipment. To solve these problems, we developed a simple and rapid MV-NAb detection system using lentivirus-based virus-like particles incorporated with the NanoLuc fragment peptide HiBiT comprising the MV fusion protein and hemagglutinin on their exterior surface. Overall, this simple, safe, and rapid method could be used to detect MV NAbs.

Establishment of measles virus receptor-expressing Vero cells lacking functional poliovirus receptors.

Global efforts are underway to eliminate measles and rubella, and active viral surveillance is the key to achieving this goal. In addition, the World Health Organization announced guidelines for handling materials potentially infectious for poliovirus (PV) to minimize the risk of PV reintroduction and to achieve PV eradication. To support global efforts, we established new PV-non-susceptible cell lines that are useful for the isolation of measles virus (MeV) and rubella virus (RuV) (Vero ΔPVR1/2 hSLAM+). In the cell lines, MeV and RuV replicated efficiently, with no concern regarding PV replication.

Poliovirus-nonsusceptible Vero cell line for the World Health Organization global action plan.

Polio or poliomyelitis is a disabling and life-threatening disease caused by poliovirus (PV). As a consequence of global polio vaccination efforts, wild PV serotypes 2 and 3 have been eradicated around the world, and wild PV serotype 1-transmitted cases have been largely eliminated except for limited regions. However, vaccine-derived PV, pathogenically reverted live PV vaccine strains, has become a serious issue. For the global eradication of polio, the World Health Organization is conducting the third edition of the Global Action Plan, which is requesting stringent control of potentially PV-infected materials. To facilitate the mission, we generated a PV-nonsusceptible Vero cell subline, which may serve as an ideal replacement of standard Vero cells to isolate emerging/re-emerging viruses without the risk of generating PV-infected materials.

Photocontrollable mononegaviruses.

Mononegaviruses are promising tools as oncolytic vectors and transgene delivery vectors for gene therapy and regenerative medicine. By using the Magnet proteins, which reversibly heterodimerize upon blue light illumination, photocontrollable mononegaviruses (measles and rabies viruses) were generated. The Magnet proteins were inserted into the flexible domain of viral polymerase, and viruses showed strong replication and oncolytic activities only when the viral polymerases were activated by blue light illumination.

A chicken homologue of nectin-4 functions as a measles virus receptor.

Measles virus (MV) vaccine strains use CD46, signaling lymphocyte activation molecule, and nectin-4 in human cells as receptors. Meanwhile, many of them are propagated in primary chicken embryonic fibroblasts (CEFs). Our data revealed that CEFs express a nectin-4 homologous molecule (CEF nectin-4) containing well-conserved motifs in the FG and BC loops, but not in the C'C″ loop. MV infected CHO cells expressing CEF nectin-4 and induced syncytia in these cells, confirming that CEF nectin-4 functions as an MV receptor and that the C'C″ loop is not critical for this function. Nectin-4-blind mutations in MV H protein reduced the infectivity of MV in CEF nectin-4-expressing cells. Infection of CEFs with the MV vaccine AIK-C strain was partially blocked by an anti-nectin-4 antibody, indicating that CEF nectin-4 plays a role for propagation of MV vaccines in CEFs.

Postinfection passive transfer of KD-247 protects against simian/human immunodeficiency virus-induced CD4+ T-cell loss in macaque lymphoid tissue.

BACKGROUND: Preadministration of high-affinity humanized anti-HIV-1 mAb KD-247 by passive transfer provides sterile protection of monkeys from heterologous chimeric simian/human immunodeficiency virus infection. METHODS: Beginning 1 h, 1 day, or 1 week after simian/human immunodeficiency virus-C2/1 challenge (20 50% tissue culture infective dose), mature, male cynomolgus monkeys received multiple passive transfers of KD-247 (45 mg/kg) on a weekly basis for approximately 2 months. Concentrations and viral loads were measured in peripheral blood, and CD4 T-cell counts were examined in both peripheral blood and various lymphoid tissues. RESULTS: Pharmacokinetic examination revealed similar plasma maintenance levels ranging from 200 to 500 microg/ml of KD-247 in the three groups. One of the six monkeys given KD-247 could not maintain these concentrations, and elicitation of anti-KD-247 idiotype antibody was suggested. All monkeys given KD-247 exhibited striking postinfection protection against both CD4 T-cell loss in various lymphoid tissues and atrophic changes in organs compared with control group animals treated with normal human immunoglobulin G. The KD-247-treated groups were also partially protected against plasma viral load elevation in peripheral blood samples, although the complete protection previously reported with preadministration of this mAb was not achieved. CONCLUSION: Postinfection passive transfer of humanized mAb KD-247 with strong neutralizing capacity against challenged virus simian/human immunodeficiency virus-C2/1 protected CD4 T cells in lymphoid organs.

Experimental evaluation of a new antithrombogenic stent using ion beam surface modification.

A new antithrombogenic stent using ion beam surface modification nanotechnology was evaluated. The ion stent is being developed to inhibit acute and chronic stent-related thrombosis. Thirty self-expanding mesh stents were fabricated from Ti-Ni metal wires with a dimension of 4 mm (diameter) x 25 mm (length) x 0.15 mm (thickness). Twenty stents were coated with type I collagen and irradiated with a He(+) ion beam at an energy of 150 keV with fluences of 1 x 10(14) ions/cm(2) (ion stent group). Ten stents had no treatment (non-ion stent group). The self-expanding stents were implanted into the right and left peripheral femoral arteries of 15 beagle dogs (vessel diameter approximately 3 mm) via a 6Fr catheter under fluoroscopic guidance. Heparin (100 units/kg) was administered intravenously before implantation. Following stent implantation, no antiplatelet or anticoagulant drugs were administered. The 1-month patency rate for the non-ion stent group was 10% (1/10), and for the ion stent group it was 80% (16/20) with no anticoagulant or antiplatelet drugs given after stent implantation (P = 0.0004 by Fisher's exact test). Ten stents remain patent after 2 years in vivo with no anticoagulant or antiplatelet drugs. These results indicate that He(+) ion-implanted collagen-coated Ti-Ni self-expanding stents have excellent antithrombogenicity and biocompatibility. This ion stent is promising for coronary and cerebral stent applications.

Chimeric adenovirus type 5/35 vector encoding SIV gag and HIV env genes affords protective immunity against the simian/human immunodeficiency virus in monkeys.

Replication-defective adenovirus type 5 (Ad5) vector-based vaccines are widely known to induce strong immunity against immunodeficiency viruses. To exploit this immunogenicity while overcoming the potential problem of preexisting immunity against human adenoviruses type 5, we developed a recombinant chimeric adenovirus type 5 with type 35 fiber vector (rAd5/35). We initially produced a simian immunodeficiency virus (SIV) gag DNA plasmid (rDNA-Gag), a human immunodeficiency virus type 1 (HIV-1) 89.6 env DNA plasmid (rDNA-Env) and a recombinant Ad5/35 vector encoding the SIV gag and HIV env gene (rAd5/35-Gag and rAd5/35-Env). Prime-boost vaccination with rDNA-Gag and -Env followed by high doses of rAd5/35-Gag and -Env elicited higher levels of cellular immune responses than did rDNAs or rAd5/35s alone. When challenged with a pathogenic simian human immunodeficiency virus (SHIV), animals receiving a prime-boost regimen or rAd5/35s alone maintained a higher number of CD4(+) T cells and remarkably suppressed plasma viral RNA loads. These findings suggest the clinical promise of an rAd5/35 vector-based vaccine.

Recombinant vaccinia DIs expressing simian immunodeficiency virus gag and pol in mammalian cells induces efficient cellular immunity as a safe immunodeficiency virus vaccine candidate.

A highly attenuated vaccinia virus substrain of Dairen-I (DIs) shows promise as a candidate vector for eliciting positive immunity against immune deficiency virus. DIs was randomly obtained by serial 1-day egg passages of a chorioarantoic membrane-adapted Dairen strain (DIE), resulting in substantial genomic deletion, including various genes regulating the virus-host-range. To investigate the impact of that deletion and of the subsequent insertion of a foreign gene into that region of DIs on the ability of the DIs recombinant to induce antigen-specific immunity, we generated a recombinant vaccinia DIs expressing fulllength gag and pol genes of simian immunodeficiency virus (SIV) (rDIsSIV gag/pol) and studied the biological and immunological characteristics of the recombinant natural mutant. The rDIsSIV gag/pol developed a tiny plaque on the chick embryo fibroblast (CEF). Viral particles of rDIsSIV gag/pol as well as SIV Gag-like particles were electromicroscopically detected in the cytoplasm. Interestingly, the recombinant DIs strain grows well in CEF cells but not in mammalian cells. While rDIsSIV gag/pol produces SIV proteins in mammalian HeLa and CV-1 cells, recombinant modified vaccinia Ankara strain (MVA) expressing SIV gag and pol genes (MVA/SIV239 gag/pol) clearly replicates in HeLa and CV-1 cell lines under synchronized growth conditions and produces the SIV protein in all cell lines. Moreover, intradermal administration of rDIsSIV gag/pol or of MVA/SIV239 gag/pol elicited similar levels of IFN-gamma spot-forming cells specific for SIV Gag. If the non-productive infection characteristically induced by recombinant DIs is sufficient to trigger immune induction, as we believe it is, then a human immunodeficiency virus vaccine employing the DIs recombinant would have the twin advantages of being both effective and safe.

Anti-V3 humanized antibody KD-247 effectively suppresses ex vivo generation of human immunodeficiency virus type 1 and affords sterile protection of monkeys against a heterologous simian/human immunodeficiency virus infection.

In an accompanying report (Y. Eda, M. Takizawa, T. Murakami, H. Maeda, K. Kimachi, H. Yonemura, S. Koyanagi, K. Shiosaki, H. Higuchi, K. Makizumi, T. Nakashima, K. Osatomi, S. Tokiyoshi, S. Matsushita, N. Yamamoto, and M. Honda, J. Virol. 80:5552-5562, 2006), we discuss our production of a high-affinity humanized monoclonal antibody, KD-247, by sequential immunization with V3 peptides derived from human immunodeficiency virus type 1 (HIV-1) clade B primary isolates. Epitope mapping revealed that KD-247 recognized the Pro-Gly-Arg V3 tip sequence conserved in HIV-1 clade B isolates. In this study, we further demonstrate that in vitro, KD-247 efficiently neutralizes CXCR4- and CCR5-tropic primary HIV-1 clade B and clade B' with matching neutralization sequence motifs but does not neutralize sequence-mismatched clade B and clade E isolates. Monkeys were provided sterile protection against heterologous simian/human immunodeficiency virus challenge by the passive transfer of a single high dose (45 mg per kg of body weight) of KD-247 and afforded partial protection by lower antibody doses (30 and 15 mg per kg). Protective neutralization endpoint titers in plasma at the time of virus challenge were 1:160 in animals passively transferred with a high dose of the antibody. The antiviral efficacy of the antibody was further confirmed by its suppression of the ex vivo generation of primary HIV-1 quasispecies in peripheral blood mononuclear cell cultures from HIV-infected individuals. Therefore, KD-247 promises to be a valuable tool not only as a passive immunization antibody for the prevention of HIV infection but also as an immunotherapy for the suppression of HIV in phenotype-matched HIV-infected individuals.

Induction of positive cellular and humoral immune responses by a prime-boost vaccine encoded with simian immunodeficiency virus gag/pol.

It is believed likely that immune responses are responsible for controlling viral load and infection. In this study, when macaques were primed with plasmid DNA encoding SIV gag and pol genes (SIVgag/pol DNA) and then boosted with replication-deficient vaccinia virus DIs recombinant expressing the same genes (rDIsSIVgag/pol), this prime-boost regimen generated higher levels of Gag-specific CD4+ and CD8+ T cell responses than did either SIVgag/pol DNA or rDIsSIVgag/pol alone. When the macaques were i.v. challenged with pathogenic simian/HIV, the prime-boost group maintained high CD4+ T cell counts and reduced plasma viral loads up to 30 wk after viral challenge, whereas the rDIsSIVgag/pol group showed only a partial attenuation of the viral infection, and the group immunized with SIVgag/pol DNA alone showed none at all. The protection levels were better correlated with the levels of virus-specific T cell responses than the levels of neutralization Ab responses. These results demonstrate that a vaccine regimen that primes with DNA and then boosts with a replication-defective vaccinia virus DIs generates anti-SIV immunity, suggesting that it will be a promising vaccine regimen for HIV-1 vaccine development.

Priming-boosting vaccination with recombinant Mycobacterium bovis bacillus Calmette-Guérin and a nonreplicating vaccinia virus recombinant leads to long-lasting and effective immunity.

Virus-specific T-cell responses can limit immunodeficiency virus type 1 (HIV-1) transmission and prevent disease progression and so could serve as the basis for an affordable, safe, and effective vaccine in humans. To assess their potential for a vaccine, we used Mycobacterium bovis bacillus Calmette-Guérin (BCG)-Tokyo and a replication-deficient vaccinia virus strain (DIs) as vectors to express full-length gag from simian immunodeficiency viruses (SIVs) (rBCG-SIVgag and rDIsSIVgag). Cynomolgus macaques were vaccinated with either rBCG-SIVgag dermally as a single modality or in combination with rDIsSIVgag intravenously. When cynomologus macaques were primed with rBCG-SIVgag and then boosted with rDIsSIVgag, high levels of gamma interferon (IFN-gamma) spot-forming cells specific for SIV Gag were induced. This combination regimen elicited effective protective immunity against mucosal challenge with pathogenic simian-human immunodeficiency virus for the 1 year the macaques were under observation. Antigen-specific intracellular IFN-gamma activity was similarly induced in each of the macaques with the priming-boosting regimen. Other groups receiving the opposite combination or the single-modality vaccines were not effectively protected. These results suggest that a recombinant M. bovis BCG-based vector may have potential as an HIV/AIDS vaccine when administered in combination with a replication-deficient vaccinia virus DIs vector in a priming-boosting strategy.

Vaccination of rhesus macaques with recombinant Mycobacterium bovis bacillus Calmette-Guérin Env V3 elicits neutralizing antibody-mediated protection against simian-human immunodeficiency virus with a homologous but not a heterologous V3 motif.

Although the correlates of vaccine-induced protection against human immunodeficiency virus type 1 (HIV-1) are not fully known, it is presumed that neutralizing antibodies (NAb) play a role in controlling virus infection. In this study, we examined immune responses elicited in rhesus macaques following vaccination with recombinant Mycobacterium bovis bacillus Calmette-Guérin expressing an HIV-1 Env V3 antigen (rBCG Env V3). We also determined the effect of vaccination on protection against challenge with either a simian-human immunodeficiency virus (SHIV-MN) or a highly pathogenic SHIV strain (SHIV-89.6PD). Immunization with rBCG Env V3 elicited significant levels of NAb for the 24 weeks tested that were predominantly HIV-1 type specific. Sera from the immunized macaques neutralized primary HIV-1 isolates in vitro, including HIV-1BZ167/X4, HIV-1SF2/X4, HIV-1CI2/X4, and, to a lesser extent, HIV-1MNp/X4, all of which contain a V3 sequence homologous to that of rBCG Env V3. In contrast, neutralization was not observed against HIV-1SF33/X4, which has a heterologous V3 sequence, nor was it found against primary HIV-1 R5 isolates from either clade A or B. Furthermore, the viral load in the vaccinated macaques was significantly reduced following low-dose challenge with SHIV-MN, and early plasma viremia was markedly decreased after high-dose SHIV-MN challenge. In contrast, replication of pathogenic SHIV-89.6PD was not affected by vaccination in any of the macaques. Thus, we have shown that immunization with an rBCG Env V3 vaccine elicits a strong, type-specific V3 NAb response in rhesus macaques. While this response was not sufficient to provide protection against a pathogenic SHIV challenge, it was able to significantly reduce the viral load in macaques following challenge with a nonpathogenic SHIV. These observations suggest that rBCG vectors have the potential to deliver an appropriate virus immunogen for desirable immune elicitations.

A consecutive priming-boosting vaccination of mice with simian immunodeficiency virus (SIV) gag/pol DNA and recombinant vaccinia virus strain DIs elicits effective anti-SIV immunity.

To evaluate immunity induced by a novel DNA prime-boost regimen, we constructed a DNA plasmid encoding the gag and pol genes from simian immunodeficiency virus (SIV) (SIVgag/pol DNA), in addition to a replication-deficient vaccinia virus strain DIs recombinant expressing SIV gag and pol genes (rDIsSIVgag/pol). In mice, priming with SIVgag/pol DNA, followed by rDIsSIVgag/pol induced an SIV-specific lymphoproliferative response that was mediated by a CD4+-T-lymphocyte subset. Immunization with either vaccine alone was insufficient to induce high levels of proliferation or Th1 responses in the animals. The prime-boost regimen also induced SIV Gag-specific cellular responses based on gamma interferon secretion, as well as cytotoxic-T-lymphocyte responses. Thus, the regimen of DNA priming and recombinant DIs boosting induced Th1-type cell-mediated immunity, which was associated with resistance to viral challenge with wild-type vaccinia virus expressing SIVgag/pol, suggesting that this new regimen may hold promise as a safe and effective vaccine against human immunodeficiency virus type 1.

Hemagglutinating virus of Japan protein is efficient for induction of CD4+ T-cell response by a hepatitis B core particle-based HIV vaccine.

By using the hepatitis B core (HBc) protein gene as a carrier, HIV-1 env V3 gene was inserted into the carrier gene, and the HIV gene was expressed inside a chimeric HIV-HBc particle (HIV-HBc), which was a unique candidate for induction of HIV-specific CTL activity. This was seen significantly in mice without the need of an adjuvant, because other responses specific for the HIV peptide such as T-cell proliferation and antibody production were not induced. However, when hemagglutinating virus of Japan (HVJ) protein was incorporated into an anionic liposome containing HIV peptide (HIV-HVJ-liposome) and was used as a booster immunization in HIV-HBc primed animals, the HIV-specific T-cell response and enhanced CTL activity were clearly induced in consecutively immunized animals. Furthermore, the HIV-specific humoral immune response was also induced and a neutralization activity was detected in the immune sera. Thus, when an HIV peptide antigen is expressed inside the virus like a particle of HBc, it can induce both cellular and humoral immunities when an HVJ-HIV-liposome, but not an HIV-liposome, is inoculated as the booster antigen. The HVJ-stimulated splenocytes secreted IL-18 and IL-12 to synergistically enhance the secretion of IFN-gamma in vitro. These findings suggest that the HVJ protein is effective at inducing the HIV-specific immunities, if used as part of a booster antigen in the consecutive immunization regimen.

Intravenous inoculation of replication-deficient recombinant vaccinia virus DIs expressing simian immunodeficiency virus gag controls highly pathogenic simian-human immunodeficiency virus in monkeys.

To be effective, a vaccine against human immunodeficiency virus type 1 (HIV-1) must induce virus-specific T-cell responses and it must be safe for use in humans. To address these issues, we developed a recombinant vaccinia virus DIs vaccine (rDIsSIVGag), which is nonreplicative in mammalian cells and expresses the full-length gag gene of simian immunodeficiency virus (SIV). Intravenous inoculation of 10(6) PFU of rDIsSIVGag in cynomologus macaques induced significant levels of gamma interferon (IFN-gamma) spot-forming cells (SFC) specific for SIV Gag. Antigen-specific lymphocyte proliferative responses were also induced and were temporally associated with the peak of IFN-gamma SFC activity in each macaque. In contrast, macaques immunized with a vector control (rDIsLacZ) showed no significant induction of antigen-specific immune responses. After challenge with a highly pathogenic simian-human immunodeficiency virus (SHIV), CD4(+) T lymphocytes were maintained in the peripheral blood and lymphoid tissues of the immunized macaques. The viral set point in plasma was also reduced in these animals, which may be related to the enhancement of virus-specific intracellular IFN-gamma(+) CD8(+) cell numbers and increased antibody titers after SHIV challenge. These results demonstrate that recombinant DIs has potential for use as an HIV/AIDS vaccine.

HIV mucosal vaccine: nasal immunization with gp160-encapsulated hemagglutinating virus of Japan-liposome induces antigen-specific CTLs and neutralizing antibody responses.

Nasal immunization of normal mice with HIVgp160-encapsulated hemagglutinating virus of Japan (HVJ)-liposome induced high titers of gp160-specific neutralizing IgG in serum and IgA in nasal wash, saliva, fecal extract, and vaginal wash, along with both Th1- and Th2-type responses. HIVgp160-specific IgG- and IgA-producing cells were also detected in mononuclear cells isolated from spleen, nasal cavity, salivary gland, intestinal lamina propria, and vaginal tissue of nasally immunized mice. In addition, CD8(+) CTLs were induced in mice nasally immunized with gp160-HVJ-liposome. These findings suggest that two layers of effective HIV-specific humoral and cellular immunity, in mucosal and systemic sites, were induced by this nasal vaccine. In immunodeficient mice, nasal immunization with gp160-HVJ-liposome induced Ag-specific immune responses for the systemic and mucosal compartments of both Th1 (IFN-gamma(-/-)) and Th2 (IL-4(-/-)). In vitro Ag-specific serum IgG Ab and vaginal wash samples possessing IgA and IgG Abs that had been induced by nasal immunization with gp160-HVJ-liposome were able to neutralize a clinically isolated strain of HIV-MN strain isolated from Japanese hemophiliac patients. Taken together, these results suggest that, for the prevention and control of AIDS, nasally administered gp160-HVJ-liposome is a powerful immunization tool that induces necessary Ag-specific immune responses at different stages of HIV infection.

Structural analysis of vaccinia virus DIs strain: application as a new replication-deficient viral vector.

DIs is a restrictive host range mutant of vaccinia virus strain DIE that grows well only in chick embryo fibroblast cells but is unable to grow in most mammalian cells. In this study, we identified one major deletion (15.4 kbp) which results in the loss of 19 putative open reading frames in the left end of the genome. We then established a system to express foreign genes by inserting them into the deleted region of DIs. We constructed rDIs to express the bacteriophage T7 polymerase (T7pol) gene and showed the expression in various mammalian cell lines by reporter luciferase gene expression under the T7 promoter. We also expressed the full-length human immunodeficiency virus (HIV)-1 NL432 gag gene. The expressed gag gene product induced high levels of cytotoxic T lymphocytes in immunized mice. These data suggest that DIs is useful as an efficient, transient replication-deficient viral vector.