Cross-protection of mice against a global spectrum of rabies virus variants.

Rabies, a continuing worldwide problem, kills tens of thousands of people and millions of animals each year. The problem is most severe in developing countries, where cell culture-derived vaccines are unaffordable and the available nervous tissue-derived vaccines are often of questionable immunogenicity and may produce neurological complications. To determine the feasibility of developing a vaccine with worldwide applicability, we investigated whether recombinant vaccinia viruses expressing either the glycoprotein (G), the nucleoprotein (N), or both the G and N (GN) of the challenge virus strain (CVS) of rabies virus would cross-protect mice against 17 rabies virus isolates representing the spectrum of rabies virus variants found worldwide. The results were compared with the commercially available human diploid cell vaccine (HDCV). Among mice injected with any of the 17 viruses, > or = 95% were protected by vaccination with recombinant viruses expressing G or GN, and > or = 85% of the mice were protected by the HDCV. The recombinant virus expressing N was less protective, protecting against only 11 of the 17 viruses. Antibody prepared against the G of the strains used in the vaccines neutralized all 17 viruses, and sera from mice infected with any one virus variant cross-neutralized all of the other viruses. Thus, no antigenic differences that would potentiate vaccine failures were identified. These studies suggest that a single rabies virus strain or its G would protect globally against wild-type rabies viruses.     

Progress towards rabies control.

Although safe and efficacious tissue-culture-derived rabies vaccines are available in developed countries, much of the world still depends on vaccines derived from neural tissue which were introduced half a century ago. Considerable advances have been made in our understanding of the molecular biology of rabies virus, and genetically engineered recombinant viruses (vaccinia-rabies virus glycoprotein) have been developed. These may facilitate the control of rabies in some species by oral vaccination campaigns.     

家养犬、猫及野生动物中狂犬病病毒流行病学调查及我国不同类型狂犬病疫苗免疫效果观测研究

选择我国狂犬病高发地区(湖南、贵州)、低发地区(江苏、武汉)和无狂犬病报告地区(沈阳)等三种不同类型的地区,开展针对家养犬、猫和啮齿类动物,以及蝙蝠等野生动物的狂犬病病毒带毒率的流行病学调查,对分离到的病毒株在抗原型别、基因变异以及与现行疫苗是否匹配等方面进行分析、比较研究。结果显示:我国狂犬病的主要宿主动物为犬,捕获犬总带毒率为2.56%;犬中狂犬病病毒的监测点最高阳性检出率达到20.0%;啮齿类动物及蝙蝠等野生动物在本次研究未检测到狂犬病病毒。我国自主研制成功的以aG株和CTN株为毒种的现行人用狂犬病疫苗,经应用研究发现,CTN株的糖蛋白基因与本研究分离到的街毒核酸序列更接近。为进一步评价现行疫苗的有效性,在5个观察点分别以aG株和CTN株毒种生产的疫苗接种两组人群(每组各约50人),结果显示均未出现中、强度反应,疫苗免疫后第7d和14d产生的中和抗体分别达到0.49IU/mL~0.52IU/mL和6.7IU/mL~7.53IU/mL;抗体阳转率分别为45.1%~47.9%和100%,表明这些疫苗具有良好的安全性和保护效果。     

A sensitive in vitro assay for the detection of residual viable rabies virus in inactivated rabies vaccines

Rabies is a viral disease transmitted through bites from rabid animals and can be prevented by vaccines. Clinically used rabies vaccines are prepared from inactivated rabies viruses grown in cell cultures or embryonated eggs. In Japan and across the world, tests that confirm complete inactivation, such as the in vivo suckling mouse assay, in which suckling mice are intracerebrally inoculated with vaccine products, are required for quality control. In this study, we developed a novel cell-based immunofluorescence assay that does not require mice for testing rabies vaccine inactivation for human use. The sensitivity of this cell-based in vitro assay was 5.7 times that of the in vivo suckling mouse assay, with a detection limit of one focus forming units per ml of test sample. This newly developed in vitro assay may replace the established in vivo suckling mouse assay for confirming viral vaccine inactivation.     

Vaccinia and other poxvirus expression vectors.

Over the past year improvements have been made in recombinant vaccinia virus gene expression and a new method for inserting DNA into the poxvirus genome has been developed, along with alternative methods for selecting recombinant viruses. Attenuated and non-replicating vaccinia virus and avian poxvirus vectors are now being used successfully. Field trials of an oral, wild-life rabies vaccine and phase 1 testing of human vaccines derived from vaccinia virus are underway.     

Creation of DNA vaccine vector based on codon-optimized gene of rabies virus glycoprotein (G protein) with consensus amino acid sequence

An optimized design of the rabies virus glycoprotein (G protein) for use within DNA vaccines has been suggested. The design represents a territorially adapted antigen constructed taking into account glycoprotein amino acid sequences of the rabies viruses registered in the Russian Federation and the vaccine Vnukovo-32 strain. Based on the created consensus amino acid sequence, the nucleotide codon-optimized sequence of this modified glycoprotein was obtained and cloned into the pVAX1 plasmid (a vector of the last generation used in the creation of DNA vaccines). A twofold increase in this gene expression compared to the expression of the Vnukovo-32 strain viral glycoprotein gene in a similar vector was registered in the transfected cell culture. It has been demonstrated that the accumulation of modified G protein exceeds the number of the control protein synthesized using the plasmid with the Vnukovo-32 strain viral glycoprotein gene by 20 times. Thus, the obtained modified rabies virus glycoprotein can be considered to be a promising DNA vaccine antigen.     

B Cell Infection and Activation by Rabies Virus-Based Vaccines

Replication-deficient rabies viruses (RABV) are promising rabies postexposure vaccines due to their prompt and potent stimulation of protective virus neutralizing antibody titers, which are produced in mice by both T-dependent and T-independent mechanisms. To promote such early and robust B cell stimulation, we hypothesized that live RABV-based vaccines directly infect B cells, thereby activating a large pool of antigen-presenting cells (APCs) capable of providing early priming and costimulation to CD4⁺ T cells. In this report, we show that live RABV-based vaccine vectors efficiently infect naive primary murine and human B cells ex vivo. Infection of B cells resulted in the significant upregulation of early markers of B cell activation and antigen presentation, including CD69, major histocompatibility complex class II (MHC-II), and CD40 in murine B cells or HLA-DR and CD40 in human B cells compared to mock-infected cells or cells treated with an inactivated RABV-based vaccine. Furthermore, primary B cells infected with a live RABV expressing ovalbumin were able to prime and stimulate naive CD4⁺ OT-II T cells to proliferate and to secrete interleukin-2 (IL-2), demonstrating a functional consequence of B cell infection and activation by live RABV-based vaccine vectors. We propose that this direct B cell stimulation by live RABV-based vaccines is a potential mechanism underlying their induction of early protective T cell-dependent B cell responses, and that designing live RABV-based vaccines to infect and activate B cells represents a promising strategy to develop a single-dose postexposure rabies vaccine where the generation of early protective antibody titers is critical.     

New vaccines for immunization of man: new approaches towards the prevention of rabies in man

Cell culture rabies vaccines for human use, highly immunogenic and well tolerated, are now used for pre-exposure immunization as well as for post-exposure treatment. Presently available cell culture rabies vaccines induce immunity against the SAD modified live rabies virus used for oral immunization of foxes. They also induce immunity against the newly identified European bat rabies virus (Duvenhage).     

In-Depth Characterization of Live Vaccines Used in Europe for Oral Rabies Vaccination of Wildlife

Although rabies incidence has fallen sharply over the past decades in Europe, the disease is still present in Eastern Europe. Oral rabies immunization of wild animal rabies has been shown to be the most effective method for the control and elimination of rabies. All rabies vaccines used in Europe are modified live virus vaccines based on the Street Alabama Dufferin (SAD) strain isolated from a naturally-infected dog in 1935. Because of the potential safety risk of a live virus which could revert to virulence, the genetic composition of three commercial attenuated live rabies vaccines was investigated in two independent laboratories using next genome sequencing. This study is the first one reporting on the diversity of variants in oral rabies vaccines as well as the presence of a mix of at least two different variants in all tested batches. The results demonstrate the need for vaccine producers to use new robust methodologies in the context of their routine vaccine quality controls prior to market release.     

Molecular Epidemiology of Rabies Viruses Circulating in Two Rabies Endemic Provinces of Laos, 2011–2012: Regional Diversity in Southeast Asia

BackgroundAlthough rabies is endemic in Laos, genetic characterization of the viruses in this country is limited. There are growing concerns that development in the region may have increased transport of dog through Laos for regional dog meat consumption, and that this may cause spillover of the viruses from dogs brought here from other countries. This study was therefore undertaken to evaluate the current rabies situation and the genetic characteristics of rabies viruses currently circulating in Laos.MethodsWe determined the rate of rabies-positive samples by analyzing data from animal samples submitted to the Lao Ministry of Agriculture and Forestry’s National Animal Health Centre rabies laboratory from 2004 through 2011. Twenty-three rabies-positive samples were used for viral genetic characterization. Full genome sequencing was performed on two rabies viruses.ResultsRabies-positive samples increased substantially from 40.5% in 2004 to 60.2% in 2009 and continued at this level during the study period. More than 99% of the samples were from dogs, followed by cats and monkeys. Phylogenetic analyses showed that three rabies virus lineages belonging to the Southeast Asian cluster are currently circulating in Laos; these are closely related to viruses from Thailand, Cambodia and Vietnam. Lineages of the circulating Laos rabies viruses diverged from common ancestors as recently as 44.2 years and as much as 55.3 years ago, indicating periodic virus invasions.ConclusionThere is an increasing trend of rabies in Laotian animals. Similar to other rabies-endemic countries, dogs are the main viral reservoir. Three viral lineages closely related to viruses from neighboring countries are currently circulating in Laos. Data provide evidence of periodic historic exchanges of the viruses with neighboring countries, but no recent invasion.     

中国19个狂犬病病毒街毒分离株N基因的序列分析

测定了30年来从不同动物中分离的19个中国狂犬病病毒街毒株N基因的部分核酸序列,并对其核苷酸差异做了比较分析.可将中国狂犬病病毒街毒株分为4个组群,各组间的同源性为83.45%～88.62%.除广西地区分离的狂犬病病毒街毒株彼此差异较大外,其余街毒株的地理分布与其N基因核酸序列差异的距离是密切相关的,基本上可按其地理分布分为东、西二大组.     

双抗体夹心ELISA与NIH法在狂犬病疫苗抗原检测中的比较

通过用单克隆抗体制备双抗体夹心ELISA,快速检测狂犬病疫苗中狂犬病毒糖蛋白(G)的含量,将狂犬病疫 苗的检定时间由28天缩短至2个工作日,以此缩短疫苗库存待检时间,提高疫苗的生产和销售效率,并最终替代 NIH动物法。将待检的狂犬病疫苗样品在同一性别12～14g昆明鼠体内作效力检定试验(NIH),同时用双抗体夹 心ELISA检测狂犬病疫苗中狂犬病毒G蛋白含量,用Microsoft Office Excel做出标准品和各样品疫苗的线性关 系图并计算出疫苗效力值E-NIH。结果用双抗体夹心ELISA所得的E-NIH与对应的小鼠效力试验所得结果M- NIH之间呈正相关性;同一批狂犬病疫苗分次测得E-NIH值在2．41～5．85之间,而相应的M-NIH值在5．11～ 10．19之间。从而得出E-NIH与相应的M-NIH之间存在明显的线性关系;与NIH法比较,ELISA具有重复性好、 成本低、快速等优点;用双抗体夹心ELISA替代小鼠效力试验是可能并可行的。     

Molecular epidemiology of rabies epizootics in Colombia, 1994-2002: evidence of human and canine rabies associated with chiroptera

Three urban rabies outbreaks have been reported in Colombia during the last two decades, one of which is ongoing in the Caribbean region (northern Colombia). The earlier outbreaks occurred almost simultaneously in Arauca (eastern Colombia) and in the Central region, ending in 1997. Phylogenetic relationships among rabies viruses isolated from the three areas were based on a comparison of cDNA fragments coding for the endodomain of protein G and a fragment of L protein obtained by RT-PCR. The sequenced amplicons which included the G-L intergenic region contained 902 base pairs. Phylogenetic analysis showed three distinct groups of viruses. Colombian genetic variant I viruses were isolated only from Arauca and the Central region, but are now apparently extinct. Colombian genetic variant II viruses were isolated in the Caribbean region and are still being transmitted in that area. The third group of bat rabies variants were isolated from two insectivorous bats, three domestic dogs and a human. This associates bat rabies virus with rabies in Colombian dogs and humans, and indicates bats to be a rabies reservoir of public health significance.     

Sensitive Procedure for Detecting Residual Viable Virus in Inactivated Rabies Vaccine

A procedure for testing inactivated rabies vaccines of tissue culture origin for residual viable virus is reported in which the vaccine to be tested is passed in primary hamster kidney cell culture (PHK) before mouse inoculation. In preliminary experiments, titrations of rabies virus in which each dilution was passed in PHK before inoculating mice yielded titers 100 to 10,000 times higher than the titers obtained for the same virus by direct mouse inoculation. This rabies virus amplification procedure was evaluated by testing 18 lots of inactivated rabies vaccine of tissue culture origin. No viable virus was found in these vaccine lots when tested by direct intracerebral inoculation of mice. Eight of these 18 lots were found to contain viable virus, however, when tested by passage in PHK cell culture. The significance of low levels of viable virus in rabies vaccines is discussed. It is recommended that the amplification procedure described in this report be used in the safety testing of rabies vaccines of tissue culture origin and that it be evaluated for use in testing other rabies vaccines of low tissue content.     

Novel Vaccines to Human Rabies

Rabies, the most fatal of all infectious diseases, remains a major public health problem in developing countries, claiming the lives of an estimated 55,000 people each year. Most fatal rabies cases, with more than half of them in children, result from dog bites and occur among low-income families in Southeast Asia and Africa. Safe and efficacious vaccines are available to prevent rabies. However, they have to be given repeatedly, three times for pre-exposure vaccination and four to five times for post-exposure prophylaxis (PEP). In cases of severe exposure, a regimen of vaccine combined with a rabies immunoglobulin (RIG) preparation is required. The high incidence of fatal rabies is linked to a lack of knowledge on the appropriate treatment of bite wounds, lack of access to costly PEP, and failure to follow up with repeat immunizations. New, more immunogenic but less costly rabies virus vaccines are needed to reduce the toll of rabies on human lives. A preventative vaccine used for the immunization of children, especially those in high incidence countries, would be expected to lower fatality rates. Such a vaccine would have to be inexpensive, safe, and provide sustained protection, preferably after a single dose. Novel regimens are also needed for PEP to reduce the need for the already scarce and costly RIG and to reduce the number of vaccine doses to one or two. In this review, the pipeline of new rabies vaccines that are in pre-clinical testing is provided and an opinion on those that might be best suited as potential replacements for the currently used vaccines is offered.     

Evaluation of the single radial-immunodiffusion assay for measuring the glycoprotein content of rabies vaccines

The glycoprotein content of rabies vaccines containing the Pitman-Moore strain of rabies virus was measured by the single radial immunodiffusion assay and correlated with vaccine potency. The variability of this assay was 6.3% for a single vaccine lot tested over a one-year period. Using sera prepared against rabies virus glycoprotein from different strains of virus, the assay gave different values. These differences could be eliminated by using a homologous vaccine strain as an internal reference. Single radial-immunodiffusion values for Pitman-Moore vaccines correlated with the manufacturers' NIH potency assay, but required a mathematical transformation to convert values from one assay to the other. Single radial-immunodiffusion values for Street Alabama Dufferin and Flury-LEP vaccines did not correlate with NIH values. Modification of the single radial immunodiffusion technique and the feasibility of using this assay for the determination of rabies vaccine potency are discussed.     

Rabies Group-Specific Ribonucleoprotein Antigen and a Test System for Grouping and Typing of Rhabdoviruses

Cell-associated ribonucleoprotein (RNP) was isolated from BHK-21 cells infected with several strains of rabies and rabies-related viruses. The RNP-antigen from rabies and related viruses induced the formation of complement-fixing, precipitating, and immunofluorescent antibodies, and proved to be the group-specific antigen common to all rabies viruses. Antigens of the envelope which induce virus-neutralizing antibodies are apparently determinative for the serotype of a virus as evidenced by two-way neutralization tests. A combination of these methods seems to be a useful approach to the serological grouping and typing of rhabdoviruses.     

The effect of strain differences on the assay of rabies virus glycoprotein by single radial immunodiffusion

Antigenic differences between rabies virus strains used for vaccine manufacture can be demonstrated using monoclonal antibodies. We have shown that these differences are sufficiently large to affect the potency values of vaccines measured in single radial immunodiffusion (SRD) assays if the reference and test vaccines are antigenically heterologous. The production of reagents for use in SRD assays for each strain of rabies virus should be considered.     

Rabies virus infects mouse and human lymphocytes and induces apoptosis.

Attenuated and highly neurovirulent rabies virus strains have distinct cellular tropisms. Highly neurovirulent strains such as the challenge virus standard (CVS) are highly neurotropic, whereas the attenuated strain ERA also infects nonneuronal cells. We report that both rabies virus strains infect activated murine lymphocytes and the human lymphoblastoid Jurkat T-cell line in vitro. The lymphocytes are more permissive to the attenuated ERA rabies virus strain than to the CVS strain in both cases. We also report that in contrast to that of the CVS strain, ERA viral replication induces apoptosis of infected Jurkat T cells, and cell death is concomitant with viral glycoprotein expression, suggesting that this protein has a role in the induction of apoptosis. Our data indicate that (i) rabies virus infects lymphocytes, (ii) lymphocyte infection with the attenuated rabies virus strain causes apoptosis, and (iii) apoptosis does not hinder rabies virus production. In contrast to CVS, ERA rabies virus and other attenuated rabies virus vaccines stimulate a strong immune response and are efficient live vaccines. The paradoxical finding that a rabies virus triggers a strong immune response despite the fact that it infects lymphocytes and induces apoptosis is discussed in terms of the function of apoptosis in the immune response.     

Oral vaccination in the control of feral rabies

The authors briefly report the results of laboratory and epidemiological investigations on living modified and inactivated antirabies vaccines, started in 1975 and carried out in collaboration with public health authorities and scientific institutions. The antirabies oral vaccination of foxes, using a live and modified vaccine (SADB19 Tüb.) began in Brescia province (Val Camonica) in 1984 and was extended in 1985 to Bolzano and Trento provinces. Since July 1986 no more cases of rabies have been observed in Italy. The problems related to the distribution in the territory of live and modified antirabies vaccines, the immunological value of inactivated vaccines, the connections between sylvatic rabies and bat rabies (or pseudorabies), are discussed.