Identification and characterization of a human monoclonal antibody that potently neutralizes a broad panel of rabies virus isolates

Rabies is a zoonosis that results in millions of human exposures worldwide each year. Human monoclonal antibodies (HuMAbs) that neutralize rabies virus may represent one viable strategy for post-exposure prophylaxis in humans, and have many advantages over current human or equine rabies immune globulin. Transgenic mice carrying human immunoglobulin genes were used to isolate human monoclonal antibodies that neutralized rabies virus. Several HuMAbs were identified that neutralized rabies virus variants from a broad panel of isolates of public health significance. HuMAb 17C7 was the most promising antibody identified because it neutralized all rabies virus isolates tested. HuMAb 17C7 recognizes a conformational epitope on the rabies virus glycoprotein which includes antigenic site III. HuMAb 17C7 protected hamsters from a lethal dose of rabies virus in a well-established in vivo model of post-exposure prophylaxis.     

Vaccination challenge studies with variants of street rabies virus isolated in Nigeria

In a preliminary study it was observed that adult ICR mice immunized with serial dilutions of an inactivated experimental human rabies vaccine from the Pitman-Moore (PM) vaccine virus were well protected against challenge with homologous virulent PM virus and challenge virus standard (CVS). However only one of five variant representatives in five of seven groups of 41 isolates of street rabies virus from Nigeria characterized by hybridoma monoclonal antibodies specific for the nucleocapsid and glycoprotein antigens of rabies virion was protected for by the vaccine. Guinea pigs immunized with a live attenuated low egg passage (LEP, Flury strain) vaccine currently used in canine vaccination in Nigeria protected against challenge with all five variants. The LEP vaccine protected against the variants and CVS quite well even when 1:125 dilution of the vaccine was used.     

Efficacy of rabies vaccines against Duvenhage virus isolated from European house bats (Eptesicus serotinus), classic rabies and rabies-related viruses

Isolates of rabies from separate enzootics can be distinguished by their reactions with panels of monoclonal antibodies (mAbs) directed to different sites on the nucleocapsid and glycoproteins of the virus. Estimates of antigenic relatedness can be made by comparing similarities among groups. In this manner it can be shown that while classic strains of rabies react with most of the mAbs, the rabies related Lyssaviruses (Mokola, Lagos and Duvenhage) react with only a few of the mAbs and isolates of rabies from Eptesicus serotinus bats in Europe are intermediate between the two groups. Mice immunized intraperitoneally with human diploid vaccine (HDCV) or animal vaccines (Rabisin and Rabiffa) were protected against a challenge with DBV, DUV-1 and most classic rabies strains. HDCV gave only partial protection against human virus isolates from Finland and Saudi Arabia. The HDCV did not protect mice against challenges with Lagos bat or Mokola virus (rabies-like viruses). The animal vaccines, however, did protect mice against Lagos bat virus, but not against Mokola. Dogs immunized with Rabisin were protected against an intracerebral challenge with DBV. Dogs developed rabies-neutralizing antibody titres after intramuscular or intravenous inoculation with live DBV or DUV-1 virus; these dogs were protected against an intramuscular canine street rabies virus challenge. We conclude that the rabies vaccines tested protect against DBV/DUV-1 and classic street rabies strains, but not Mokola.     

Bat-transmitted human rabies outbreaks, Brazilian Amazon

We describe 2 bat-transmitted outbreaks in remote, rural areas of Portel and Viseu Municipalities, Pará State, northern Brazil. Central nervous system specimens were taken after patients' deaths and underwent immunofluorescent assay and histopathologic examination for rabies antigens; also, specimens were injected intracerebrally into suckling mice in an attempt to isolate the virus. Strains obtained were antigenically and genetically characterized. Twenty-one persons died due to paralytic rabies in the 2 municipalities. Ten rabies virus strains were isolated from human specimens; 2 other cases were diagnosed by histopathologic examination. Isolates were antigenically characterized as Desmodus rotundus variant 3 (AgV3). DNA sequencing of 6 strains showed that they were genetically close to D. rotundus-related strains isolated in Brazil. The genetic results were similar to those obtained by using monoclonal antibodies and support the conclusion that the isolates studied belong to the same rabies cycle, the virus variants found in the vampire bat D. rotundus.     

Epidemiologic analysis of antigenic variations of street rabies virus: detection by monoclonal antibodies

The nucleocapsid antigen of 204 strains of street rabies virus, which originated in Europe, Africa and Asia, was analyzed by fluorescent antibody staining technique with a panel of 20 monoclonal antibodies specific for nucleocapsid of rabies and rabies-related viruses. A definite pattern of reactivity was observed with strains of the same geographic origin with the exception of strains originating from Madagascar, Thailand and Iran which were more diversified. Mice immunized with a vaccine prepared from a Pasteur PV-11 strain of virus were well protected against challenge with representative strains from Europe and Africa, and a partial protection was observed following challenge with strains from Madagascar and Thailand.     

Bat rabies, Texas, 1996-2000

Bats submitted to the Texas Department of Health (1996-2000) were speciated and tested for rabies virus antigen by direct immunofluorescence microscopy. Antigenic analysis of rabies virus-positive specimens was performed with monoclonal antibodies against the nucleoprotein of the virus; atypical or unexpected results were confirmed by genetic analysis of nucleoprotein sequence.     

Molecular and antigenic characterization of rabies viruses from Iran identifies variants with distinct epidemiological origins

A molecular epidemiological study of 48 recent rabies isolates recovered from cases reported throughout Iran identified three distinct viral variants, the evolutionary origins of which were identified by phylogenetic comparison with rabies viruses originating from Europe and Asia. Members of group 1 (15 isolates) were recovered from the northern half of the country only, while those of group 2 (31 isolates) were widely dispersed; both groups clustered within the widely disseminated cosmopolitan lineage. The two isolates of group 3 were detected in the northeastern tip of the country only and belonged to the Arctic strain. Rapid variant discrimination tools, employing restriction fragment length polymorphisms applied to amplified fragments of the viral genome, were devised whilst antigenic characterization of representative viruses identified a small panel of monoclonal antibodies that were also discriminatory. The future application of such methods should provide valuable epidemiological information on rabies incidence in Iran.     

Genetic and antigenic analysis of street strains of rabies virus in GuangxiP.R.China

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广西狂犬病毒野毒株的基因和抗原分析（英文）

Analyse the genetic and antigenic variant of rabies virus in Guangxi with epidemiological method, monoclonal antibodies reaction pattern and molecular biological technique. Rabies incidence has increase in recent three years in Guangxi, especially in mountainous areas. Investigation into the human rabies found that there are about 20％ vaccinated defeat cases.On the basis of their reactivity to monoclonal antibodies against the viral nucleocapsid protein(mAb-N),4 street strains isolated from different areas where enzootic rabies is shown had different reaction patterns compared with Chinese vaccine strain(3aG). The nucleotide and amino acid sequence of street strain(GX89_1) G and N gene were compared with 3aG, the overall nucleotide homology of GX89_1 with 3aG was 84.5％ and amino acid homology of GX89_1 with 3aG was 89.5％ in G gene.The overall nucleotide homology of GX89_1 with 3aG was 86％ in N gene and they were not in the same group.There are several amino acid replacement in AA 243_268 of G protein that can affect the antigenicity of rabies virus.The findings suggest that there are different street strains of rabies virus in Guangxi.     

The protective role of humoral neutralizing antibody in the NIH potency test for rabies vaccines

Intraperitoneal vaccination of mice with rabies vaccine results in both dosage-dependent rabies virus neutralizing antibody titres and protection from lethal intracerebral (i.c.) challenge with fixed strain CVS rabies virus. Pre-exposure adoptive intravenous transfer of naive or immune cells did not significantly protect naive Balb/c mice from lethal i.c. CVS challenge, but immune serum and anti-rabies glycoprotein monoclonal antibodies (individually and in combination) did confer significant protection when administered before or up to 24 h after lethal i.c. rabies virus challenge.     

湖南省武岗市洞口县狂犬病流行病学研究

目的分析2003-2004年间在湖南省武岗市与洞口县发生的狂犬病并探讨局部地区流行的因素.方法对狂犬病进行个案调查,用直接免疫荧光法检测犬脑组织中的狂犬病毒抗原,用RT-PCR法扩增N基因片段,测定核苷酸序列并构建系统发生树进行遗传特征分析.结果自1991-2002年武岗市与洞口县仅各报告1例人狂犬病,但2003-2004年间,分别报告30例人狂犬病.62例狂犬病患者中61例被犬所伤,1例被猫所伤.在有完整记录的50例患者中,平均潜伏期为44.18天.其中7例（14%）狂犬病患者的潜伏期≤15天,16例（32%）潜伏期≤20天.在疫点周围采集的99只犬脑组织中,13只犬脑组织狂犬病毒抗原阳性,阳性率为13.13%.将Wg13与Dk13株病毒的部分N基因核苷酸序列与已报道中国病毒株比较,发现2株病毒与广西及安徽的毒株有最高的同源性,构建的系统发生树也分在同一分支.用全N基因核苷酸序列分析发现,Wg13与Dk13毒株全为Ⅰ型狂犬病毒,2株病毒之间的同源性为99.4%以上.比较分析2株病毒N基因编码的氨基酸序列,发现包括第Ⅳ抗原位点区在内的多个氨基酸位点发生了氨基酸的替代.结论引起武岗市与洞口县的狂犬病流行的病毒不是新型狂犬病毒,犬饲养量大与病毒携带率高是狂犬病暴发的直接原因.     

Is there an advantage to including the nucleoprotein in a rabies glycoprotein subunit vaccine?

The PV rabies (genotype 1) G and N proteins were produced by recombinant baculoviruses in insect cells. We tested the ability of recombinant antigens to synergistically induce an immune response and, particularly, to broaden the spectrum of Lyssavirus-neutralizing antibodies produced. Cell-free preparations of recombinant proteins caused an immune response. Recombinant rabies G protein (RRG) from infected cell extract or supernatant induced virus neutralizing antibodies (VNAb) against rabies (CVS virus (genotype 1) and in a less extent against European Bat Lyssavirus-1 (EBL-1:genotype 5) Recombinant rabies N protein (RRN) induced antibodies that reacted with the rabies virus ribonucleoprotein (RNP) and primed mice for both the production of VNAb induced by inactivated and purified rabies virus and the protection conferred by RNP. RRN also had an adjuvant effect on VNAb production induced by RRG when the two recombinant proteins were physically associated either encapsulated in liposomes or subjected to ultrasound treatment. However, there was no increase in production of VNAb directed against EBL-1 although classical vaccines (genotype 1) induce partial protection against this virus. thus, beside its adjuvant effect there is some doubt as to whether including rabies N protein in a rabies subunit vaccine containing the recombinant G protein would be advantageous.     

Rabies virus-like particles expressed in HEK293 cells

Rabies is an infectious viral disease with a mortality rate close to 100%. Currently, there is a need to generate cheaper and more immunogenic vaccines for the effective prevention of rabies, mostly in developing countries. Virus-like particles have been widely used in viral vaccine production due to their high immunogenicity and safety during the production process. Rabies virus glycoprotein is the major antigen to trigger a protective immune response and the only protein capable of generating virus neutralizing antibodies. In this study we describe the development of a recombinant stable cell line for the production of rabies virus-like particles (RV-VLPs) expressing the rabies virus glycoprotein by lentivirus-based transduction of HEK293 cells. Protein expression was analyzed by flow cytometry, fluorescence microscopy, western blot and ELISA. Particles were purified from culture supernatant and their size and morphology were studied. Furthermore, mice were immunized with RV-VLPs, formulated with adjuvant, and these particles were able to produce a specific antibody response, demonstrating that these virus-like particles present a promising rabies vaccine candidate.     

Inhibition of immune responses against rabies virus by monoclonal antibodies directed against rabies virus antigens.

Treatment of mice with a cocktail of murine anti-rabies monoclonal antibodies (mAb-C) interfered with the ability of these animals to mount a virus-neutralizing antibody response to rabies vaccine. Administered mAb-C did not affect the induction of rabies virus-specific T-helper cells. The magnitude of the inhibition of rabies virus-specific B-cell response was dependent on the concentration of the mAb-C and the duration of the mAb-mediated interference was inversely proportional to the biological half-life of the mAb. As long as the serum titres were above a critical threshold, the suppression could not be overcome even by multiple vaccinations. Since injection of mice with immunocomplexes consisting of inactivated rabies virus and mAb rendered the animals non-responsive to a subsequent vaccination with inactivated rabies virus, it is concluded that the mAb-induced suppression might be caused by the formation of antigen-antibody complexes which exert a negative signalling effect to premature B cells.     

Rabies virus strains circulating in Bhutan: implications for control

We report a molecular epidemiological study of rabies virus (RABV) strains circulating in animal populations in Bhutan, and investigate potential origins of these viruses. Twenty-three RABV isolates originating from dogs and other domestic animals were characterized by sequencing the partial nucleoprotein (N) gene (395 bp). Phylogenetic analysis was conducted and the Bhutanese isolates were compared with rabies viruses originating from other parts of the world. Phylogenetic analysis showed that Bhutanese isolates were highly similar and were closely related to Indian strains and South Asian Arctic-like-1 viruses. Our study suggests that the rabies viruses spreading in southern parts of Bhutan have originated from a common ancestor, perhaps from the Indian virus strain.     

Expression in plants and immunogenicity of plant virus-based experimental rabies vaccine

A new approach to the production and delivery of vaccine antigens is the use of engineered amino virus-based vectors. A chimeric peptide containing antigenic determinants from rabies virus glycoprotein (G protein) (amino acids 253-275) and nucleoprotein (N protein) (amino acids 404-418) was PCR-amplified and cloned as a translational fusion product with the alfalfa mosaic virus (AlMV) coat protein (CP). This recombinant CP was expressed in two plant virus-based expression systems. The first one utilized transgenic Nicotiana tabacum cv. Samsun NN plants providing replicative functions in trans for full-length infectious RNA3 of AlMV (NF1-g24). The second one utilized Nicotiana benthamiana and spinach (Spinacia oleracea) plants using autonomously replicating tobacco mosaic virus (TMV) lacking native CP (Av/A4-g24). Recombinant virus containing the chimeric rabies virus epitope was isolated from infected transgenic N. tabacum cv. Samsun NN plants and used for parenteral immunization of mice. Mice immunized with recombinant virus were protected against challenge infection. Based on the previously demonstrated efficacy of this plant virus-based experimental rabies vaccine when orally administered to mice in virus-infected unprocessed raw spinach leaves, we assessed its efficacy in human volunteers. Three of five volunteers who had previously been immunized against rabies virus with a conventional vaccine specifically responded against the peptide antigen after ingesting spinach leaves infected with the recombinant virus. When rabies virus non-immune individuals were fed the same material, 5/9 demonstrated significant antibody responses to either rabies virus or AlMV. Following a single dose of conventional rabies virus vaccine, three of these individuals showed detectable levels of rabies virus-neutralizing antibodies, whereas none of five controls revealed these antibodies. These findings provide clear indication of the potential of the plant virus-based expression systems as supplementary oral booster for rabies vaccinations.     

Phylogenetic analysis of nucleoprotein gene of dog rabies virus isolates from Southern India

India like several other South East Asian and African countries continues to face the public health and economic problems associated with the disease. Our objective was to perform a limited sequence analysis of a portion of nucleoprotein gene of 22 rabies virus isolates obtained from domestic animals in Southern India during 2004–2005. These isolates were compared with rabies virus isolates originating from Asia, Europe, Africa and North America. The phylogenetic analysis showed that RV isolates in Southern India belong to genotype 1. They were similar to one another forming a single major genetic cluster not ordered by geography or species of origin. However, they were dissimilar to RV isolates in Northern India and in other parts of the world. The data indicated that dog rabies virus variants are the major circulating viruses and control of dog rabies would result in overall reduction in the burden and incidence of rabies in India.     

Evaluation of a new serological technique for detecting rabies virus antibodies following vaccination

Two major techniques are currently used to estimate rabies virus antibody values: neutralization assays, such as the rapid fluorescent focus inhibition test (RFFIT), and enzyme-linked immunosorbent assays (ELISAs). The RFFIT is considered the gold standard assay and has been used to assess the titer of rabies virus neutralizing antibodies for more than three decades. In the late 1970s, ELISA began to be used to estimate the level of rabies virus antibody and has recently been used by some laboratories as an alternate screening test for animal sera. Although the ELISA appears simpler, safer and more efficient, the assay is less sensitive in detecting low values of rabies virus neutralizing antibodies than neutralization tests. This study was designed to evaluate a new ELISA-based method for detecting rabies virus binding antibody. This new technique uses electro-chemi-luminescence labels and carbon electrode plates to detect binding events. In this comparative study, the RFFIT and the new ELISA-based technique were used to evaluate the level of rabies virus antibodies in human and animal serum samples. By using a conservative approximation of 0.15 IU/ml as a cutoff point, the new ELISA-based technique demonstrated a sensitivity of 100% and a specificity of 95% for human samples and for experimental animal samples. The sensitivity and specificity for field animal samples was 96% and 95%, respectively. The preliminary results from this study appear promising and demonstrate a higher sensitivity than traditional ELISA methods.     

Rabies virus isolates of India – Simultaneous existence of two distinct evolutionary lineages

Rabies is a fatal viral disease of serious public health implication. The disease is enzootic in India. In the present study, thirty six rabies virus isolates were obtained from terrestrial mammals of India during 2002–2012. Ecto-domain coding region of the glycoprotein gene from all the isolates were sequenced and the phylogenetic analysis was performed in relation to the global rabies and rabies related virus isolates. The Indian isolates grouped into two distinctly separate lineages with majority of the Indian isolates in Arctic like 1 lineage and the remaining isolates in sub-continental lineage. Isolates of the two distinct lineages were identified simultaneously from the same geographical region. Time scaled phylogenetic tree indicated that the sub-continental lineage of the virus is one of the earliest clade of rabies virus that diverged from bat rabies virus. On the contrary, the Arctic-like 1 lineage of India appeared to be a more recent divergence event. The amino acid sequence comparison revealed that all the major antigenic sites were almost conserved among the Indian isolates whereas few amino acid variations could be identified around site IIa, minor site I and IV. The d_N/d_S study based on G ecto-domain is in support of the earlier reports of strong purifying selection. In conclusion, it is evident that the Indian rabies virus isolates are of two major distinct lineages with distant phylogenetic and evolutionary relationship.