Oral vaccination of captive arctic foxes with lyophilized SAG2 rabies vaccine

Arctic foxes (Alopex lagopus) were immunized with lyophilized SAG2 oral rabies vaccine. The effectiveness of this vaccine was determined by serologic response and survival to challenge by rabies virus isolated from a red fox from Alaska (USA). No vaccine virus was found in saliva 1-72 hr after ingestion. At 2 wk after vaccination, all foxes had seroconverted, with rabies virus neutralizing antibody levels of 0.2-3.1 IU ml(-1). All vaccinated foxes survived to week 17 after challenge, and hippocampus, pons, and cerebellum were free of rabies virus as determined by direct immunofluorescence testing after death. One of four nonvaccinated foxes survived challenge and was free of rabies virus in neural tissue, and no rabies virus neutralizing antibody was detected in blood. Our results suggest that the lyophilized SAG2 oral rabies vaccine could be effective in arctic and subarctic regions, where freezing air and ground temperatures probably would not reduce its immunogenicity.     

Rabies-related viruses

Five viruses related to rabies occur in Africa. Two of these, Obodhiang from Sudan and kotonkan from Nigeria, were found in insects and are only distantly related to rabies virus. The other three are antigenically more closely related to rabies. Mokola virus was isolated from shrews in Nigeria, Lagos bat virus from fruit bats in Nigeria, and Duvenhage virus from brain of a man bitten by a bat in South Africa. The public health significance of the rabies-related viruses was emphasized in Zimbabwe where in 1981 a rabies-related virus became epizootic in the dog and cat population. It is postulated that the ancestral origin of rabies virus was Africa where the greatest antigenic diversity occurs and that the ancestor may have been an insect virus. Questions are raised why rabies has not evolved more rapidly in the New World, given the frequency and ease with which antigenic changes can be induced in the laboratory, and how the virus became so extensively established in New World bats.     

人源抗狂犬病毒单克隆抗体Fab段基因的获得和表达

运用噬菌体表面呈现（phage display）技术获得了人源抗狂犬病毒糖蛋白基因工程单克隆抗体Fab段基因及其表达。从狂犬病毒PM株Vero细胞疫苗免疫的人抗凝血中分离获得外周淋巴细胞,提取细胞总RNA,通过RTPCR方法,用一组人IgG Fab基因4特异性引物,从合成的cDNA中扩增了一组轻链和重链Fab段基因,将轻链和重链Fab段基因,将轻链和重链先后克隆入噬菌体载体pComb3,成功地建立了抗狂犬病毒抗原的方法,对此抗体库进行富积筛选表达,成功地获得了抗狂犬病毒的人源单抗Fab段基因及其在大肠杆菌中的有效表达,对其中一株单抗G10进行了较为系统的分析,发现它与一株鼠源中和性狂犬病毒糖蛋白特异性单抗存在竞争,证实该单抗能识别狂犬病毒糖蛋白,其序列资料分析表明,该单抗为一株新的抗狂犬病毒人源基因工程抗体。     

Experimental inoculation of raccoons (Procyon lotor) with rabies virus of skunk origin.

To determine raccoon (Procyon lotor) susceptibility and serum neutralizing antibody response to a skunk salivary gland rabies virus, raccoons were inoculated with a rabies virus isolated from a naturally-infected striped skunk (Mephitis mephitis). Raccoons were divided into four groups of three animals each. A dilution of the rabies virus suspension, 10(2.4), 10(3.4), or 10(4.8), mouse intracerebral lethal dose50 (MICLD50), was administered into the masseter muscles of each animal. Three negative control animals received only diluent. Saliva and sera were collected on post-inoculation days 35, 63 and 92 for virus isolation and determination of serum neutralizing antibody titer. All animals survived the 92 day observation period and none exhibited the behavioral changes classically associated with clinical rabies virus infections. Rabies virus was not detected in the saliva of any raccoon and two of the three animals receiving the highest inoculum developed serum neutralizing antibodies (SNA). On day 92, a challenge suspension of New York City/Georgia (NYC/GA) strain rabies virus in fox salivary glands (10(3.2) MICLD50) was administered to all 12 raccoons. All animals succumbed to rabies virus except the two animals that had earlier developed SNA. The results of this study provided evidence about the susceptibility of raccoons to a skunk rabies virus and demonstrated that exposed raccoons could survive for at least 92 days following exposure. Furthermore, animals developing SNA under such circumstances were capable of withstanding challenge with rabies virus that was fatal for seronegative raccoons.     

Isolation and characterization of human T cell lines and clones reactive to rabies virus: antigen specificity and production of interferon-gamma

By using a preparation of inactivated rabies virus, the blood mononuclear cells from five rabies vaccine recipients were stimulated in vitro in the presence of interleukin 2. T cell lines that displayed significant proliferative responses to whole rabies virus and to preparations of rabies glycoprotein and nucleocapsid were obtained from all the individuals. Other antigens, such as diphtheria and tetanus toxoids, influenza A virus, hepatitis B surface antigen, and serum albumin, failed to induce the proliferation of the T cell lines. One of these rabies-specific T cell lines was found to proliferate in response to rabies antigens only when the antigen-presenting cells expressed homologous HLA-DR antigens. The use of mouse monoclonal antibodies specific for human T cell surface markers revealed that most of the cells of these rabies-reactive lines were of the helper/inducer class of T lymphocytes. Stimulation of the T cell lines with the rabies antigens induced the production of interferon-gamma, a lymphokine with potent antiviral activity. Several T cell clones were isolated from two of these cell lines, and most of them appeared to be specific for the antigenic components of the viral nucleocapsid. Two T cell clones specific for the rabies glycoprotein were also isolated from one of these lymphocyte interleukin 2-dependent lines. Further in vitro studies with rabies-specific T cells could help us to understand in more depth the role of regulatory T cells in the human immune response to rabies virus.     

Evidence for prenatal transfer of rabies virus in the Mexican free-tailed bat (Tadarida brasiliensis Mexicana)

Fetuses were collected from four Mexican free-tailed bats (Tadarida brasiliensis mexicana) and a fetal bat cell (FBC) line was established and tested for its ability to support the replication of the ERA vaccine strain of rabies virus. Cytopathic effects were detected in ERA virus-inoculated as well as uninoculated FBC's. Immunofluorescent antibody testing of uninoculated FBC's provided no evidence for the presence of rabies virus. However, mice inoculated intracranially with supernatant fluid from uninoculated FBC's died. Enzyme-linked immunosorbent assay and immunofluorescent antibody testing revealed rabies virus in the brains of these mice. Tests with a panel of monoclonal antibodies indicated that the isolate was the same as that isolated from Mexican free-tailed bats from the southwestern United States. We conclude that the fetuses from which the FBC line was derived had been infected in utero with rabies virus. We believe this may represent the first observation of prenatal transfer of rabies virus in naturally infected bats.     

Whole-genome sequencing and phylogenetic analysis of rabies viruses from Jordan

Human fatalities caused by rabies are rarely reported in Jordan; however, domestic animals are more likely to fall victim to rabies compared to wild animals, at least this is the case in Jordan due to the presence of canine rabies. In this study, twelve brain samples from domestic and wild animals suspected of being infected with rabies virus from different regions of Jordan were collected during 2019. Seven of them tested positive using the fluorescent antibody test and real-time SYBR RT-PCR assay. Five specimens were from stray dogs and two from foxes. The whole genome sequences were obtained from the positive samples. Sequence analysis showed that one dog virus from Al Quwaysimah city located in Amman governorate, was closely related to an Israeli strain belonging to a Cosmopolitan ME1a clade. The genomes of the remaining six viruses (four from dogs and two from foxes) collected from different areas of Jordan were genetically-related to each other and clustered together with sequences from Iran and Turkey; all belong to Cosmopolitan ME2 clade. These sequences were analyzed with six other Jordanian rabies virus nucleoprotein (N) gene sequences available in the public database, five of them belong to ME1a clade and one belongs to ME1b clade. Rabies virus whole genome data is scarce across the Middle East. This study provides a better understanding of the molecular epidemiology of rabies virus in the region.     

狂犬病毒糖蛋白DNA疫苗的研制及其免疫效果的观察

构建了含有狂犬病毒（RV）CVS株糖蛋白（GP）基因的重组质粒pCMVCVSRG,将其转染至鼠NIH3T3细胞中,用间接免疫荧光法和APAAP法均证实RVGP能在真核细胞中表达。分别将合RV不同毒株的GP基因的质粒（DNA疫苗）及空白载体质粒（对照组）免疫小鼠,仅DNA疫苗免疫的小鼠产生了中和抗体。以RV攻击后,DNA疫苗免疫组小鼠的存活率与对照组相比,差异有极显著性意义（P＜0．01）;不同的启动子（CMV或SV40）与不同GP基因（来源于CVS株或ERA株）对DNA疫苗的免疫效果无明显影响。在注射120d后．用PCR方法仍可检测出RVGP基因。结果表明：狂犬病DNA疫苗能够诱生低水平的中和抗体和记忆性B淋巴细胞,并能保护小鼠抵抗RV的攻击。该疫苗能在体内稳定存在。狂犬病DNA疫苗的研制为狂犬病免疫开辟了一条新途径,并可为防治其他疾病的DNA疫苗的研制奠定基础。     

The Production of Antibody by Invading B Cells Is Required for the Clearance of Rabies Virus from the Central Nervous System

Background

The pathogenesis of rabies is associated with the inability to deliver immune effectors across the blood-brain barrier and to clear virulent rabies virus from CNS tissues. However, the mechanisms that facilitate immune effector entry into CNS tissues are induced by infection with attenuated rabies virus.

Methodology/Principal Findings

Infection of normal mice with attenuated rabies virus but not immunization with killed virus can promote the clearance of pathogenic rabies virus from the CNS. T cell activity in B cell–deficient mice can control the replication of attenuated virus in the CNS, but viral mRNA persists. Low levels of passively administered rabies virus–neutralizing antibody reach infected cells in the cerebellum of B cell–deficient mice but are not sufficient to mediate virus clearance. Production of rabies virus-specific antibody by B cells invading CNS tissues is required for this process, and a substantial proportion of the B cells that accumulate in the CNS of mice infected with attenuated rabies virus produce virus-specific antibodies.

Conclusions/Significance

The mechanisms required for immune effectors to enter rabies virus-infected tissues are induced by infection with attenuated rabies virus but not by infection with pathogenic rabies viruses or immunization with killed virus. T cell activities can inhibit rabies virus replication, but the production of rabies virus–specific antibodies by infiltrating B cells, as opposed to the leakage of circulating antibody across the BBB, is critical to elimination of the virus. These findings suggest that a pathogenic rabies virus infection may be treatable after the virus has reached the CNS tissues, providing that the appropriate immune effectors can be targeted to the infected tissues.     

Induction of rabies virus-specific T-helper cells by synthetic peptides that carry dominant T-helper cell epitopes of the viral ribonucleoprotein.

The T-helper cell response to the internal proteins of rabies virus was investigated. The rabies virus nucleoprotein was shown to be a major target antigen for T-helper cells that cross-react between rabies and rabies-related viruses. T-helper cells were assayed in vitro by testing virus-induced lymphocytes for lymphokine secretion in response to antigen. Immunodominant T-helper cell epitopes of the viral nucleoprotein were identified in vitro by using synthetic peptides delineated from the amino acid sequence of the nucleoprotein. The response to synthetic peptides were under Ir gene control. Antigenic peptides were tested in vivo for stimulation of rabies virus-specific T-helper cells. Inoculation of mice with peptides bearing immunodominant T-helper cell epitopes resulted in an accelerated and enhanced neutralizing antibody response upon booster immunization with inactivated rabies virus.     

The use of an E1-deleted, replication-defective adenovirus recombinant expressing the rabies virus glycoprotein for early vaccination of mice against rabies virus.

An E1-deleted, replication-defective adenovirus recombinant of the human strain 5 expressing the rabies virus glycoprotein, termed Adrab.gp, was tested in young mice. Mice immunized at birth with the Adrab.gp construct developed antibodies to rabies virus and cytokine-secreting lymphocytes and were protected against subsequent challenge. Maternal immunity to rabies virus strongly interferes with vaccination of the offspring with a traditional inactivated rabies virus vaccine. The immune response to the rabies virus glycoprotein, as presented by the Adrab.gp vaccine, on the other hand, was not impaired by maternal immunity. Even neonatal immunization of mice born to rabies virus-immune dams with Adrab.gp construct resulted in a long-lasting protective immune response to rabies virus, suggesting that this type of vaccine could be useful for immunization shortly after birth. Nevertheless, pups born to Adrab.gp virus-immune dams showed an impaired immune response to the rabies virus glycoprotein upon vaccination with the Adrab.gp virus, indicating that maternal immunity to the vaccine carrier affected the offspring's immune response to rabies virus.     

Isolation and replication of rabies virus in C6 rat glioma cells (clone CCL-107).

The susceptibility of the C6 rat glioma cell line (ATCC; CCL-107) to rabies virus was characterized. The kinetics of infection performed with a fixed and a wild strain (from an infected cow) of rabies virus was monitored by direct immunofluorescence. Fluorescent cytoplasmic bodies were readily observed by UV microscopy from 24 hours post-infection (hpi) onwards. The ability of C6 to produce rabies infective virion particles was confirmed by determining the viral titres present in the supernatants of infected cultures, by both BHK-21 cell infection and mice inoculation. C6 cells produced similar viral titres to those produced by BHK-21 for both strains used. In addition, the yield of rabies glycoprotein was assessed by ELISA. In general, BHK-21 and C6 cells infected either by PV or with the wild rabies strain produced similar amounts of rabies glycoprotein. At 96 hpi, however, when the glycoprotein production peaked, BHK-21 infected with the wild strain produced significantly higher amounts of glycoprotein than C6. Subsequently, the optimal conditions for isolation of wild rabies virus strains from C6 cells were established and these proved to be as sensitive as NA cells in detecting 10 wild rabies samples. Due to the high sensitivity exhibited, C6 rat glioma cells present a new and useful system for rabies virus investigation.     

Therapeutic immune clearance of rabies virus from the CNS

The long-held concept that rabies infection is lethal in humans once the causative rabies virus has reached the CNS has been called into question by the recent survival of a number of patients with clinical rabies. Studies in animal models provide insight into why survival from a rabies virus infection that has spread to the CNS is possible and the immune mechanisms involved. In the CNS, both innate mechanisms capable of inhibiting virus replication and the activity of infiltrating rabies virus-specific T and B cells with the capacity to clear the virus are required. Deficiencies in the induction of either aspect of rabies immunity can lead to lethal consequences but may be overcome by novel approaches to active and passive immunization.     

Characterization of saturable binding sites for rabies virus.

A specific, saturable receptor for rabies virus was analyzed on cultured cells of neural or non-neural origin. Viral attachment kinetics were enhanced by DEAE-dextran, an effect which in turn enhanced the apparent infectivity of the virus inoculum. Under optimized conditions, the attachment of metabolically labeled ERA strain rabies virus obeyed the laws of mass action, whereby the amount of virus bound to cells varied proportionally with the concentration of cells or virus. Attachment was sensitive to changes of temperature and pH, did not require divalent cations such as Mg2+ or Ca2+, and occurred despite prior treatment of cells with proteolytic or sialic acid-specific enzymes. Saturation of the cell surface with rabies virus could be accomplished with 3 X 10(3) to 15 X 10(3) attached virions per cell. Competition for the rabies receptor occurred with rabies nonpathogenic variant virus, RV194 -2, and vesicular stomatitis virus. Reovirus type 3, another neurotropic virus, failed to inhibit rabies virus binding, and West Nile virus only slightly inhibited rabies virus binding, suggesting independent cellular receptors were recognized by these viruses. Isolated rabies virus glycoprotein failed to compete in an equivalent manner. However, solubilization of BHK-21 cells with octylglucoside yielded a chloroform-methanol-soluble extract which blocked rabies virus attachment. The binding inhibition activity of this extract was resistant to proteases but could be destroyed by phospholipases and neuraminidase, suggesting a phospholipid or glycolipid component at the receptor site. These data provide evidence for a rhabdovirus-common mechanism for cellular attachment to cells in culture.     

Vaccination of free-ranging Pennsylvania raccoons (Procyon lotor) with inactivated rabies vaccine

Thirty-nine free-ranging raccoons (Procyon lotor) in an endemic rabies area of Pennsylvania (USA) were vaccinated with a single intramuscular inoculation of commercial inactivated rabies virus vaccine, 17 June to 23 August 1987. Paired serum samples, pre- and postvaccination, were obtained from eight raccoons and were analyzed in vitro for rabies virus neutralizing antibody using a modified rapid fluorescent focus inhibition test. Seven of eight (88%) recaptured raccoons demonstrated seroconversion within 15 to 26 days of vaccination. At 1 yr postvaccination, three vaccinated raccoons were recaptured and challenged in captivity with street rabies virus, resulting in the death of two of three vaccinates and five of five unvaccinated control raccoons.     

The influence of homologous vs. heterologous challenge virus strains on the serological test results of rabies virus neutralizing assays

The effect that the relatedness of the viral seed strain used to produce rabies vaccines has to the strain of challenge virus used to measure rabies virus neutralizing antibodies after vaccination was evaluated. Serum samples from 173 subjects vaccinated with either purified Vero cell rabies vaccine (PVRV), produced from the Pittman Moore (PM) seed strain of rabies virus, or purified chick embryo cell rabies vaccine (PCECV), produced from the Flury low egg passage (Flury-LEP) seed strain of rabies virus, were tested in parallel assays by RFFIT using a homologous and a heterologous testing system. In the homologous system, CVS-11 was used as the challenge virus in the assay to evaluate the humoral immune response in subjects vaccinated with PVRV and Flury-LEP was used for subjects vaccinated with PCECV. In the heterologous system, CVS-11 was used as the challenge virus in the assay to evaluate subjects vaccinated with PCECV and Flury-LEP was used for subjects vaccinated with PVRV. Although the difference in G protein homology between the CVS-11 and Flury-LEP rabies virus strains has been reported to be only 5.8%, the use of a homologous testing system resulted in approximately 30% higher titers for nearly two-thirds of the samples from both vaccine groups compared to a heterologous testing system. The evaluation of equivalence of the immune response after vaccination with the two different vaccines was dependent upon the type of testing system, homologous or heterologous, used to evaluate the level of rabies virus neutralizing antibodies. Equivalence between the vaccines was achieved when a homologous testing system was used but not when a heterologous testing system was used. The results of this study indicate that the strain of virus used in the biological assays to measure the level of rabies virus neutralizing antibodies after vaccination could profoundly influence the evaluation of rabies vaccines.