Comparison of an anti-rabies human monoclonal antibody combination with human polyclonal anti-rabies immune globulin

The World Health Organization estimates human mortality from endemic canine rabies to be 55,000 deaths/year. Limited supply hampers the accessibility of appropriate lifesaving treatment, particularly in areas where rabies is endemic. Anti-rabies antibodies are key to protection against lethal rabies. Currently, only human and equine polyclonal anti-rabies immune globulin (HRIG and ERIG) is available. Replacement of HRIG and ERIG with a safer and more widely available product is recommended. We have recently identified a combination of 2 human monoclonal antibodies (MAbs), CR57 and CR4098, that has high potential. We here describe a head-to-head comparison between an CR57/CR4098 MAb cocktail and HRIG. The MAb cocktail neutralized all viruses from a panel of 26 representative street rabies virus isolates. In combination with vaccine, the MAb cocktail protected Syrian hamsters against lethal rabies when administered 24 h after exposure, comparable with the results obtained with HRIG. Furthermore, the MAb cocktail did not interfere with rabies vaccine differently from HRIG. These results demonstrate that the human MAb cocktail of CR57 and CR4098 is a safe and efficacious alternative to RIG in rabies postexposure prophylaxis.     

Antibody responses to human diploid cell vaccine for rabies with and without human rabies immune globulin

One hundred one volunteers with no exposure to rabies were given human diploid cell vaccine (HDCV) for rabies with or without 20 international units of human rabies immune globulin (HRIG)/kg of body weight to evaluate schedules for therapy with HDCV and HRIG after exposure. All of the volunteers who received three or more doses of HDCV alone or four or more doses of HDCV with HRIG developed high titers of neutralizing antibodies by day 35, which persisted for at least 60 days. By day 7, of the 61 volunteers given HRIG and HDCV, 53% had neutralizing antibodies by a mouse neutralization test and 67% had neutralizing antibodies by a rapid fluorescent focus inhibition test. Similar antibody levels were found in volunteers given HRIG alone, a finding which suggests that low or undetectable early titers after administration of HDCV and HRIG were due to inadequate HRIG dosage rather than any interaction between the passive antibody (HRIG) and the vaccine antigen. These results suggest that trials with 30 or 40 international units of HRIG/kg in combination with HDCV are warranted.     

Equine rabies immune globulin: a product with an undeserved poor reputation

Four hundred nineteen patients exposed to rabies in Thailand were treated with equine rabies immune globulin (ERIG) manufactured by Sclavo of Italy, a product also licensed in the United States of America. They were followed for a minimum of 1 month after ERIG injection and rabies vaccine administration. Adverse serum sickness-like reactions were noted in 15 patients (3.58%). These were clinically acceptable and only 1 of these patients required corticosteroid therapy and short term hospitalization for serum sickness. ERIG is approximately 1/10 of the cost of human rabies immune globulin (HRIG), which is not generally available in developing countries. ERIG is a safe and underutilized essential biological when HRIG is not affordable or available.     

Isolation and Characterization of Cross-Reactive Human Monoclonal Antibodies That Potently Neutralize Australian Bat Lyssavirus Variants and Other Phylogroup 1 Lyssaviruses

Australian bat lyssavirus (ABLV) is a rhabdovirus that circulates in four species of pteropid bats (ABLVp) and the yellow-bellied sheath-tailed bat (ABLVs) in mainland Australia. In the three confirmed human cases of ABLV, rabies illness preceded fatality. As with rabies virus (RABV), post-exposure prophylaxis (PEP) for potential ABLV infections consists of wound cleansing, administration of the rabies vaccine and injection of rabies immunoglobulin (RIG) proximal to the wound. Despite the efficacy of PEP, the inaccessibility of human RIG (HRIG) in the developing world and the high immunogenicity of equine RIG (ERIG) has led to consideration of human monoclonal antibodies (hmAbs) as a passive immunization option that offers enhanced safety and specificity. Using a recombinant vesicular stomatitis virus (rVSV) expressing the glycoprotein (G) protein of ABLVs and phage display, we identified two hmAbs, A6 and F11, which completely neutralize ABLVs/ABLVp, and RABV at concentrations ranging from 0.39 and 6.25 µg/mL and 0.19 and 0.39 µg/mL respectively. A6 and F11 recognize overlapping epitopes in the lyssavirus G protein, effectively neutralizing phylogroup 1 lyssaviruses, while having little effect on phylogroup 2 and non-grouped diverse lyssaviruses. These results suggest that A6 and F11 could be effective therapeutic and diagnostic tools for phylogroup 1 lyssavirus infections.     

Diffusion and fate of intramuscularly injected human rabies immune globulin

The importance of rabies immune globulin (RIG) in postexposure rabies treatment is well known and it has been emphasized that the local injection into the animal bite sites is crucial. This preliminary study used a radioisotope tracer that allows following the fate of human rabies immune globulin (HRIG) injected intramuscularly. There was significant retention and local diffusion of the immune globulin at the injection site and significant radiotracer could still be detected at the site 24 h later.     

动物狂犬病病毒街毒株的细胞分离鉴定

目的N2A细胞和BHK-21细胞用于我国动物狂犬病流行街毒株的分离培养。方法狂犬病发病动物脑组织悬液首先脑内接种乳鼠,再采取Nonclon Delta Tube内置盖玻片法从鼠脑中进行病毒分离培养,脑组织悬液接种细胞后96h,利用直接荧光抗体实验检测盖玻片上的病毒感染细胞,判定病毒是否在细胞上增殖;通过测定不同毒株细胞培养上清中的病毒滴度对病毒进行鉴定。结果22份狂犬病病犬、猪和牛的脑组织接种乳鼠后均获得了鼠脑分离毒,将鼠脑毒接种N2A细胞分离获得了22株病毒,接种BHK-21细胞却只分离获得20株病毒。不同病毒株第三代细胞培养上清中的病毒滴度从10-1TCID50/100μL到10-3.6TCID50/100μL。结论N2A细胞对狂犬病病毒街毒株的敏感性高于BHK-21细胞,且不同的病毒株对细胞的适应能力不同。实验分离获得的病毒细胞适应株丰富了我国狂犬病病毒毒种资源,为进一步研究不同病毒分离株的抗原性差异奠定基础。     

Sequence analysis of rabies virus in humans exhibiting encephalitic or paralytic rabies

Two distinct clinical patterns, encephalitic (furious) and paralytic (dumb), have been recognized in human rabies. It has been postulated that different rabies virus variants associated with particular vectors may be responsible for these different clinical manifestations. Analysis of the glycoprotein (G), nucleoprotein (N), and phosphoprotein (P) genes of rabies viruses from 2 human cases of encephalitic rabies and from 2 human cases of paralytic rabies demonstrated only minor nucleotide differences. Deduced amino-acid patterns of the N protein were identical in both human and canine samples that came from the same geographic location, regardless of the clinical form. All differences in amino-acid patterns of the G protein were found outside the ectodomain, in either the signal peptide or the transmembrane and endodomains. None of the amino-acid differences of the P protein was within the interactive site with dynein. These findings support the concept that clinical manifestations of rabies are not explained solely by the associated rabies virus variant.     

Incidence of human rabies exposure and associated factors at the Gondar Health Center,Ethiopia: a three-year retrospective study

Background

Rabies is one of the oldest known and most feared human diseases. Epidemiological studies provide basic information about the burden of the disease and underline the importance of prevention and control interventions. However, there have been limited studies conducted regarding the incidence of rabies and associated factors in Ethiopia, in general, and in this study area, in particular. Therefore, the aim of this study was to assess the incidence of human rabies exposure and associated factors at the Gondar Health Center, Ethiopia.

Methods

A retrospective cross-sectional study was conducted at the Gondar Health Center where post-exposure prophylaxis (PEP) for rabies was available for the whole population in the North Gondar Zone catchment area. Data of human rabies exposure cases between 2011 and 2013 were collected from the rabies PEP registration book using data abstraction sheets. The data was entered and analyzed using SPSS version 16 statistical software.

Result

A total of 261 cases of human rabies exposure were reported to the Gondar Health Center from 2011 to 2013. The sex and age specific distribution showed that the majority of these cases were among males (142/226, 62.8%) and children under 15 years of age (87/226, 38.5%). A predominant number of cases were observed in individuals from rural areas (161/220, 73.2%), and during fall and winter seasons (67/222, 30.18%). A significant number of people exposed to rabies (23.2%) came to the health center for PEP two or more weeks after the injury. The incidence of human rabies exposure cases was 4.6, 2.61, and 1.27 per 100, 000 population in 2011, 2012, and 2013, respectively. Being male and living in an urban setting were found to be risk factors for human rabies exposure in 2011.

Conclusion

A significant number of human rabies exposure cases were reported to the Gondar Health Center. Being male and living in an urban setting were found to be associated with rabies exposure. A community-based follow-up study is recommended to more accurately estimate the incidence of human rabies exposure.

Electronic supplementary material

The online version of this article (doi:10.1186/2049-9957-4-3) contains supplementary material, which is available to authorized users.     

A recombinant rabies virus expressing vesicular stomatitis virus glycoprotein fails to protect against rabies virus infection

To investigate the importance of the rabies virus (RV) glycoprotein (G) in protection against rabies, we constructed a recombinant RV (rRV) in which the RV G ecto- and transmembrane domains were replaced with the corresponding regions of vesicular stomatitis virus (VSV) glycoprotein (rRV-VSV-G). We were able to recover rRV-VSV-G and found that particle production was equal to rRV. However, the budding of the chimeric virus was delayed and infectious titers were reduced 10-fold compared with the parental rRV strain containing RV G. Biochemical analysis showed equal replication rates of both viruses, and similar amounts of wild-type and chimeric G were present in the respective viral particles. Additional studies were performed to determine whether the immune response against rRV-VSV-G was sufficient to protect against rabies. Mice were primed with rRV or rRV-VSV-G and challenged with a pathogenic strain of RV 12 days later. Similar immune responses against the internal viral proteins of both viruses indicated successful infection. All mice receiving the rRV vaccine survived the challenge, whereas immunization with rRV-VSV-G did not induce protection. The results confirm the crucial role of RV G in an RV vaccine.     

狂犬病病毒弱毒疫苗株反向遗传操作系统的建立

目的构建狂犬病病毒弱毒疫苗株SRV9全长cDNA感染性克隆,并建立其反向遗传操作系统。方法通过DNAStar对狂犬病病毒SRV9株全长基因组序列进行分析,利用单一的酶切位点,将SRV9全长cDNA分为4段,根据每段重叠区域的酶切位点拼接全长,分别连入pCI和pCDNA3.1(+)载体,并通过PCR方法分别在全长序列的3′端和5′端引入核酶HamRZ和HdvRZ序列,构建全长真核表达质粒pCI-SRV9和pD-SRV9。同时构建能表达狂犬病病毒核蛋白(N)、磷蛋白(P)、糖蛋白(G)和聚合酶蛋白(L)的4个辅助质粒。分别将pCI-SRV9或pD-SRV9与辅助质粒通过脂质体共转染BSR细胞,拯救重组病毒。结果两种质粒表达系统均可拯救到有感染活性的病毒粒子,但pD-SRV9表达质粒的拯救效率(8/8)较pCI-SRV9表达质粒拯救效率3/30高;重组病毒与母本野生病毒的体外生长动力学曲线相一致,相同培养时间的病毒滴度差异无统计学意义(P>0.05)。结论成功构建了狂犬病病毒弱毒疫苗株的反向遗传操作系统,为进一步研究狂犬病病毒致病机理、筛选新型狂犬病疫苗或开发基于狂犬病病毒载体的其他疾病疫苗奠定了基础。     

抗狂犬病病毒的初步研究

目的选取盐酸氯胺酮,重组人干扰素α1b以及人狂犬病免疫球蛋白治疗带毒犬,力图为犬伤后寻找有效治疗。方法取经过盐酸氯胺酮,重组人干扰素α1b以及人狂犬病免疫球蛋白治疗的带毒犬唾液,采用快速狂犬病酶联免疫诊断(RREID),检测唾液标本中狂犬病病毒抗原转阴率分别为36.36%,7.14%,和10.00%。结果氯胺酮治疗组与干扰素及免疫球蛋白治疗组比较,病毒抗原转阴率差异有统计学意义(P<0.05)。结论盐酸氯胺酮在处理严重的犬咬伤抗狂犬病毒上具有一定效果。     

上海地区2004年疑似狂犬的病毒检测分析及其应用

目的对2004年上海地区疑似狂犬进行病毒检测,分析应用于狂犬伤多人应急事件的处理。方法收集126份疑似狂犬病的犬脑标本,用ELISA法快速检测狂犬病毒抗原、小鼠感染法(MIT)分离病毒和免疫荧光法(IF)鉴定病毒型别。对疑似狂犬进行地区和时间分布调查。结果3种方法完全一致。确诊狂犬的阳性率为22.2%(28/126),鉴定为1型狂犬病毒。6-8月检出率最高,为全年的75%(21/28)。病毒检测阳性的地区进行紧急防治措施。结论狂犬病毒检测3种方法,既可快速诊断,又可获得定型的毒株。便于对狂犬地区采取应急防治措施,利于控制预防狂犬病。     

Assessment of rabies exposure risk among Israeli travelers

BACKGROUND: The decision whether or not to administer rabies pre-exposure prophylaxis (PEP) to travelers visiting endemic areas is a complex one. Paramount for making that decision is knowledge of the risk of animal bites during travel. This study attempts to estimate the risk of bites in travelers, and study the action they took before and after the incident. METHODS: Travelers presenting for pre-travel immunizations during the period of August through December 2004, who planned a travel of >or= 1 month's duration were retrospectively identified, contacted and interviewed by a structured questionnaire. These travelers did not receive specific advice concerning rabies. RESULTS: The study cohort comprised of 815 travelers (median age=25), of who 13 (1.6%) were injured by a potentially rabid animal (mainly, dog=6; monkey=4). The incidence of potential rabies exposure was found to be of 2.66 per 1000 travelers per month. Those injured had significantly longer trips than the non-injured (6.9+/-3.8 vs. 4+/-5.0 months, p=0.037); notably, the injuries occurred after a median of 5 weeks from departure. Although seven travelers noted blood at the site of injury, only four (31%) of the injured sought medical attention following the exposure, and all four received post-exposure prophylaxis. CONCLUSIONS: An injury by potentially rabid animals is not rare among long-term travelers. As the injury may occur early in the itinerary, rabies PEP should be considered for this population. Educational efforts are required in light of the lack of understanding of the dismal consequences of rabies among travelers.     

Function and glycosylation of plant-derived antiviral monoclonal antibody

Plant genetic engineering led to the production of plant-derived mAb (mAbP), which provides a safe and economically feasible alternative to the current methods of antibody production in animal systems. In this study, the heavy and light chains of human anti-rabies mAb were expressed and assembled in planta under the control of two strong constitutive promoters. An alfalfa mosaic virus untranslated leader sequence and Lys-Asp-Glu-Leu (KDEL) endoplasmic reticulum retention signal were linked at the N and C terminus of the heavy chain, respectively. mAbP was as effective at neutralizing the activity of the rabies virus as the mammalian-derived antibody (mAbM) or human rabies Ig (HRIG). The mAbP contained mainly oligomannose type N-glycans (90%) and had no potentially antigenic alpha(1,3)-linked fucose residues. mAbP had a shorter half-life than mAbM. The mAbP was as efficient as HRIG for post-exposure prophylaxis against rabies virus in hamsters, indicating that differences in N-glycosylation do not affect the efficacy of the antibody in this model.     

A recombinant human adenovirus vaccine against rabies

Rabies continues to be a serious problem in both developed and developing nations due to the reservoir of rabies virus in wildlife vectors. The control and worldwide eradication of rabies depends on the development of safe, effective, and economical vaccines that might be used in preexposure vaccination programs for humans and animals. To this end an infectious human adenovirus type 5 recombinant virus that contains the rabies glycoprotein gene, and which may serve as the prototype for a new class of vaccines against rabies, was constructed and tested. This recombinant, when administered by either the parenteral or oronasal route, was highly effective in eliciting good levels of rabies-neutralizing antibodies in the sera of dogs and mice. Mice immunized by the recombinant virus were protected from lethal intracerebral challenge with rabies virus.     

Induction of protective immunity against rabies by immunization with rabies virus ribonucleoprotein.

We have studied the ability of rabies virus ribonucleoprotein (RNP) to induce a protective immune response in animals against lethal challenge with rabies and rabies-related lyssa viruses. Liposomes containing either RNP or the glycoprotein (G protein) of a variant virus with multiple alterations in the G antigenic structure conferred no or poor protection, respectively, against lethal intracerebral challenge with rabies virus. By contrast, liposomes containing RNP and the variant G protein induced a good protective response, comparable to that achieved with inactivated virus vaccine against intracerebral challenge. Moreover, mice or raccoons immunized with RNP alone resisted lethal peripheral challenge with homologous or heterologous virus strains. These results indicate that the RNP of rabies virus plays a crucial role in induction of protective immunity.     

Effective preexposure and postexposure prophylaxis of rabies with a highly attenuated recombinant rabies virus

Rabies remains an important public health problem with more than 95% of all human rabies cases caused by exposure to rabid dogs in areas where effective, inexpensive vaccines are unavailable. Because of their ability to induce strong innate and adaptive immune responses capable of clearing the infection from the CNS after a single immunization, live-attenuated rabies virus (RV) vaccines could be particularly useful not only for the global eradication of canine rabies but also for late-stage rabies postexposure prophylaxis of humans. To overcome concerns regarding the safety of live-attenuated RV vaccines, we developed the highly attenuated triple RV G variant, SPBAANGAS-GAS-GAS. In contrast to most attenuated recombinant RVs generated thus far, SPBAANGAS-GAS-GAS is completely nonpathogenic after intracranial infection of mice that are either developmentally immunocompromised (e.g., 5-day-old mice) or have inherited deficits in immune function (e.g., antibody production or type I IFN signaling), as well as normal adult animals. In addition, SPBAANGAS-GAS-GAS induces immune mechanisms capable of containing a CNS infection with pathogenic RV, thereby preventing lethal rabies encephalopathy. The lack of pathogenicity together with excellent immunogenicity and the capacity to deliver immune effectors to CNS tissues makes SPBAANGAS-GAS-GAS a promising vaccine candidate for both the preexposure and postexposure prophylaxis of rabies.     

Attempts to immunize sheep against Haemonchus contortus using a cocktail of recombinant proteases derived from the protective antigen,H‐gal‐GP

The objective of this study was to determine whether an antigen cocktail containing recombinantly expressed versions of most of the protective proteases of H‐gal‐GP, a known protective antigen from Haemonchus contortus, would confer any protection to lambs in a vaccine‐challenge trial. Haemonchus contortus metalloendopeptidases, MEP1, MEP3 and MEP4, were expressed as soluble recombinant proteins in insect cells, but attempts to express the H. contortus aspartyl proteases, PEP1 and PEP2, by the same techniques were not successful. Recombinant H. contortus PEP1 was therefore expressed in Escherichia coli and refolded. Groups of sheep were immunized thrice with either native H‐gal‐GP, a cocktail of recombinantly expressed proteins (rMEP1, rMEP3, rMEP4 and rPep1), or adjuvant only (QuilA in PBS). All sheep were challenged with 5000 infective larvae 1 week after the final vaccination. High levels of serum antibodies that recognized H‐gal‐GP were detected in both the native antigen and recombinant cocktail‐immunized groups by the time of challenge, but protective immunity was only observed in the group immunized with native H‐gal‐GP.     

河南省九株狂犬病毒的糖蛋白基因序列分析

目的 了解河南省狂犬病毒与人用、兽用狂犬病疫苗株在糖蛋白基因核苷酸和氨基酸序列水平上的差异,为有效控制狂犬病疫情提供初步的科学依据.方法 以反转录-半套式聚合酶链反应(RT-heminested-PCR)扩增2006年12月分离自河南省信阳市的9株狂犬病毒街毒株,经纯化、克隆、测序后获得9条糖蛋白基因全长序列,采用生物信息学软件构建基因系统发育树,对糖蛋白基因序列和氨基酸序列进行分析.结果 9株狂犬病毒糖蛋白在核苷酸及氨基酸序列水平上彼此的同源性分别为97.6%～98.9%和99.2%～99.8%;9株病毒与CTN疫苗的同源性最高,其核苷酸及氨基酸同源性分别为85.6%～93.0%和91.9%～92.9%;9株病毒与其他疫苗株相比,其核苷酸及氨基酸同源性分别为80.4%～83.3%和87.7%～92.5%;与已知的基因1型狂犬病毒比较,9株病毒糖蛋白氨基酸序列发生了若干位点的氨基酸取代.结论 9株河南省狂犬病毒街毒株均属基因1型,CTN疫苗株可能为目前我国河南省所流行的狂犬病提供较好的保护效果.     

Characterization of a unique variant of bat rabies virus responsible for newly emerging human cases in North America.

The silver-haired bat variant of rabies virus (SHBRV) has been identified as the etiological agent of a number of recent human rabies cases in the United States that are unusual in not having been associated with any known history of conventional exposure. Comparison of the different biological and biochemical properties of isolates of this virus with those of a coyote street rabies virus (COSRV) revealed that there are unique features associated with SHBRV. In vitro studies showed that, while the susceptibility of neuroblastoma cells to infection by both viruses was similar, the infectivity of SHBRV was much higher than that of COSRV in fibroblasts (BHK-21) and epithelial cells (MA-104), particularly when these cells were kept at 34 degrees C. At this temperature, low pH-dependent fusion and cell-to-cell spread of virus is seen in BHK-21 cells infected with SHBRV but not with COSRV. It appears that SHBRV may possess an unique cellular tropism and the ability to replicate at lower temperature, allowing a more effective local replication in the dermis. This hypothesis is supported by in vivo results which showed that while SHBRV is less neurovirulent than COSRV when administered via the intramuscular or intranasal routes, both viruses are equally neuroinvasive if injected intracranially or intradermally. Consistent with the above findings, the amino acid sequences of the glycoproteins of SHBRV and COSRV were found to have substantial differences, particularly in the region that contains the putative toxic loop, which are reflected in marked differences in their antigenic composition. Nevertheless, an experimental rabies vaccine based on the Pittman Moore vaccine strain protected mice equally well from lethal doses of SHBRV and COSRV, suggesting that currently used vaccines should be effective in the postexposure prophylaxis of rabies due to SHBRV.