表达狂犬病毒糖蛋白抗原的重组犬2型腺病毒的构建及鉴定

为研制一种预防犬科动物狂犬病的新型疫苗,将含有狂犬病毒ERA株糖蛋白基因(Rabies glycoprotein,Rgp)表达盒的穿梭质粒pVAXΔE3Rgp中的Rgp表达盒克隆入犬2型腺病毒(Canine adenovirus type2,CAV2)骨架质粒pPoly2-CAV2中,获得重组质粒pPoly2-CAV2-ΔE3-Rgp,释放其基因组,转染MDCK细胞系,获得E3缺失区(Deletion of early protein3,ΔE3)含有Rgp表达盒的重组病毒CAV2-ΔE3-Rgp。该重组病毒能在MDCK细胞上产生典型的腺病毒细胞病变。通过酶切、PCR、基因测序,表明该重组病毒含有完整的Rgp表达盒。通过RT-PCR、Western blot等检测,表明该重组病毒能够表达Rgp抗原。用该重组病毒免疫犬,3次免疫后,可以诱导犬产生特异的抗CAV2HI抗体,其效价超过1∶256和抗狂犬病病毒(Rabies virus,RV)中和抗体,其效价超过0.50IU/mL。试验结果表明,获得的重组病毒免疫犬后,能够产生抗狂犬病毒和腺病毒的高效价保护性抗体,是一种有潜力的犬科动物狂犬病毒腺病毒二联疫苗候选株。     

The induction of protective immune response in mice vaccinated by recombinant avian adenovirus CELO expressing glycoprotein G of the rabies virus

The recombinant avian adenovirus CELO-gpRb expressing glycoprotein G of rabies virus (strain TS-80, ARRIW&M, Pokrov, Russia) was used for mice vaccination against rabies. Double intramuscular immunization by recombinant CELO-gpRb adenovirus in a dose 10(9) pfu per mouse caused the induction of virus neutralizing antibodies (VNA) synthesis in 78% of mice, while twice repeated intradermal injections of the recombinant adenovirus failed to induce the VNA production. The protection level in groups of vaccinated mice after intracerebral injection of CVS rabies virus in a dose of 100 MLD50 was equal to 45% at single intramuscular immunization and to 91% after twice repeated intramuscular immunization. The recombinant adenoviral vaccine against rabies, based on CELO viral genome, has a good perspective for domestic and wild animal vaccination, not only due to rather high protection level, but also because the production of adenoviral CELO vaccine in chicken embryos is of high technology and inexpensive.     

Raccoon poxvirus recombinants expressing the rabies virus nucleoprotein protect mice against lethal rabies virus infection.

Raccoon poxvirus (RCN) recombinants expressing the rabies virus internal structural nucleoprotein (RCN-N) protected A/WySnJ mice against a lethal challenge with street rabies virus (SRV). Maximum survival was achieved following vaccination by tail scratch and footpad (FP) SRV challenge. RCN-N-vaccinated mice inoculated in the FP with SRV were resistant to infection for at least 54 weeks postvaccination. Protection was also elicited by RCN recombinants expressing the rabies virus glycoprotein (RCN-G). Vaccination with RCN-G evoked rabies virus neutralizing antibody. Rabies virus neutralizing antibody was not detected in RCN-N-vaccinated mice prior to or following SRV infection. Radioimmunoprecipitation assays showed that sera from RCN-N-vaccinated mice which survived SRV infection did not contain antibody to SRV structural protein G, M, or NS. The mechanism(s) of N-induced resistance appears to correlate with the failure of peripherally inoculated SRV to enter the central nervous system (CNS). Support for this correlation with resistance was documented by the observations that SRV-inoculated RCN-N-vaccinated mice did not develop clinical signs of CNS rabies virus infection, infectious SRV was not detected in the spinal cord or brain following FP challenge, and all RCN-N-vaccinated mice died following direct intracranial infection of the CNS with SRV. These results suggest that factors other than anti-G neutralizing antibody are important in resistance to rabies virus and that the N protein should be considered for incorporation with the G protein in recombinant vaccines.     

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.     

Purification of rabies virus glycoprotein produced in Drosophila melanogaster S2 cells: An efficient immunoaffinity method

Most rabies vaccines are based on inactivated virus, which production process demands a high level of biosafety structures. In the past decades, recombinant rabies virus glycoprotein (RVGP) produced in several expression systems has been extensively studied to be used as an alternative vaccine. The immunogenic characteristics of this protein depend on its correct conformation, which is present only after the correct post-translational modifications, typically performed by animal cells. The main challenge of using this protein as a vaccine candidate is to keep its trimeric conformation after the purification process. We describe here a new immunoaffinity chromatography method using a monoclonal antibody for RVGP Site II for purification of recombinant rabies virus glycoprotein expressed on the membrane of Drosophila melanogaster S2 cells. RVGP recovery achieved at least 93%, and characterization analysis showed that the main antigenic proprieties were preserved after purification.     

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.     

Inactivated rabies vaccine control and release: use of an ELISA method.

Quality control of human rabies vaccines performed by National Control Laboratories (NCLs) prior to marketing vaccines batches requires in vivo and in vitro potency assays as requested by the relevant European Pharmacopoeia monographs, OMCLs guidelines and WHO technical recommendations.The aim of the present study was to check the suitability of an enzyme-linked immunosorbent assay (ELISA) using a virus neutralizing monoclonal antibody, directed to the rabies virus glycoprotein, to monitor the consistency of the lot to lot rabies vaccines production. Furthermore, this work was implemented to establish in house specifications for the glycoprotein content.     

Standardization of an enzyme immunoassay for the in vitro potency assay of inactivated tissue culture rabies vaccines: determination of the rabies virus glycoprotein with polyclonal antisera

A non-competitive enzyme-linked immunoassay (ELISA) has been standardized to supplement the in vivo potency test used for the quality control of inactivated tissue culture vaccines against rabies. The essentials of the ELISA were: fixation of the virus in different dilutions of vaccine on the surface of microtitre plates; testing of the reference and up to six test vaccines on one plate; incubation with polyclonal antisera to rabies virus glycoprotein containing an excess of antibody; further incubation with a species-specific anti-IgG coupled to peroxidase; a final incubation with a substrate. The incubation periods were 1 h, 1 h and 30 min both at +37 degrees C. The relative potency determinations were made graphically or by a computer using a parallel line bioassay in which the potencies of the vaccines of unknown potency were tested against the reference preparation on a single microtitre plate. Under these conditions inactivated rabies vaccines of different types (virus strains, cell substrates, inactivation and concentration procedures) were tested for potency. Furthermore, it was possible with this in vitro method to assay adjuvanted vaccines, in process samples such as tissue culture supernatants with live or inactivated rabies virus, concentrates, and vaccines undergoing thermal stability tests. The rabies glycoprotein antigen-antibody reaction was highly specific according to the results and the glycoprotein content was measured quantitatively. The potency determined by the in vitro ELISA correlated with the in vivo NIH protection potency test. The lower limit of detection of the ELISA was 0.015 IU/ml. Quantitative antigen determination was possible with both homologous and heterologous antisera to rabies virus glycoprotein when vaccines of the same virus strain were tested. When the potencies of vaccines of different virus strain specificity were calculated, it was necessary to take into account the strain-specific antigenicity. Even so vaccines of high potency were found to give a stronger reaction with a heterologous serum than did weak vaccines with a homologous antiserum. Stability tests made on inactivated tissue culture vaccines such as vaccine from the human diploid cell strain (HDCS), from purified chicken embryo cell (PCEC) or from purified Vero cell rabies vaccine (PVRV), showed high stability of the glycoprotein antigen even after four months of storage at +37 degrees C or 24 h at +56 degrees C, provided that the vaccines were stored in a lyophilized state. The antigenicity of liquid vaccines was inactivated after a few hours at +56 degrees C. For tropical areas, therefore, only lyophilized vaccines should be considered.     

狂犬病病毒糖蛋白重组人腺病毒的构建及免疫效果的初步研究

构建表达狂犬病病毒弱毒SRV9糖蛋白(GP)的重组人5型腺病毒,检测其对小鼠的免疫效果。将狂犬病病毒SRV9株GP基因的完整开放阅读框克隆到腺病毒表达系统中的穿梭质粒多克隆位点,构建重组穿梭质粒pac-Ad5CMV-Gs9,以罗氏转染液介导线性化骨架质粒和重组穿梭质粒共转染293AD细胞,细胞病变后取培养物进行PCR鉴定并电镜观察,在293AD细胞上测定病毒滴度。以106 TCID50重组腺病毒腹腔接种昆明小鼠,免疫后不同时段采尾静脉血通过荧光抗体病毒中和试验(FAVN)检测小鼠血清狂犬病中和抗体效价。正确构建重组穿梭质粒pacAd5CMV-Gs9;获得表达狂犬病病毒SRV9株GP蛋白的缺陷型重组人5型腺病毒;病毒滴度达到106 CFU/mL以上;腹腔接种小鼠14d后均产生了抗狂犬病中和抗体,有效保护率达90%。成功获得了表达狂犬病病毒GP基因的重组腺病毒,该腺病毒免疫小鼠可产生保护性中和抗体,为进一步开发新型兽用狂犬病疫苗奠定了物质基础。     

Complete genome sequence of a street rabies virus from Mexico

A canine rabies virus (RABV) has been used as a street rabies virus in laboratory investigations. Its entire genome was sequenced and found to be closely related to that of canine RABV circulating in Mexico. Sequence comparison indicates that the virus is closely related to those in the "cosmopolitan" group, with high homology (89 to 93%) to clade I of rabies viruses. The virus is now termed dog rabies virus-Mexico (DRV-Mexico).     

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.     

Detection and genetic characterization of rabies virus from human patients

Saliva and blood were collected from two patients who had not received post exposure prophylaxis in the cities of Wenzhou and Xinning respectively. Both patients were confirmed as positive for rabies by detection of rabies virus specific nucleoprotein antibodies in the sera by Western Blot. However, rabies virus specific RNA was only identified in the saliva collected from the patient in Wenzhou. Furthermore, the isolate Zhejiang Wz0 (H) was obtained by inoculating one-day-old suckling mice. Both nucleoprotein (N) and glycoprotein (G) genes from the isolate were amplified by RT-PCR and sequenced. Phylogenetic analysis indicated that the isolate belonged to classic rabies virus, and shared a higher homology with the street viruses from dogs in the main endemic areas in China and the street virus from dogs in Indonesia than with other known strains. Further comparison of the deduced amino acid sequences between the isolate and the vaccine strains used in China showed that the virus had a higher level of homology with the vaccine strain CTN than with the other vaccine strains (3aG, PV, PM and ERA). In particular, amino acid residues substitutions located in antigenic site III in the G protein, which could react with the neutralizing antibodies, were observed. These results suggested that the virus belonged to the classic rabies virus, and both N and G genes diverged from the current vaccine strains used in China at either the nucleotide or the amino acid level. Foundation items: The project was supported by the Grants (2003BA712A08-02 and 2004BA718B03) from the Chinese Ministry of Science and Technology.     

Serological responses of coyotes to two commercial rabies vaccines.

Between August 1993 and September 1994 we documented serological responses of coyotes (Canis latrans) vaccinated with two commercial rabies vaccines licensed for use in domestic dogs. Serologic responses were documented by testing for rabies virus neutralizing antibodies with the rapid fluorescent focus inhibition test (RFFIT) at 30, 90, 180, 270, and 365 days post-vaccination. All coyotes vaccinated with Imrab 3 (Rhone-Merieux, Inc.), and 75% of those vaccinated with Dura-Rab 3 (Immunovet, Inc.) seroconverted, as evidenced by the presence of antirabies antibody titers > or = 1:5 in one or more of the five post-vaccination samples. The percent of coyotes showing a titer > or = 1:5 was generally greater and titer levels appeared higher and more persistent among animals vaccinated with Imrab 3 than Dura-Rab 3. Presence of titers via RFFIT tests demonstrates the antibodies produced in coyotes by these rabies vaccines functionally bind and neutralize rabies virus in vitro, but these results do not constitute a demonstration of protection required for licensure for use in coyotes.     

C-terminal lysosome targeting domain of CD63 modifies cellular localization of rabies virus glycoprotein

The glycoprotein of rabies virus is the central antigen elicited the immune response to infection; therefore, the majority of developing anti-rabies vaccines are based on this protein. In order to increase the efficacy of DNA immunogen encoding rabies virus glycoprotein, the construction of chimeric protein with the CD63 domain has been proposed. The CD63 is a transmembrane protein localized on the cell surface and in lysosomes. The lysosome targeting motif GYEVM is located at its C-terminus. We used the domain that bears this motif (c-CD63) to generate chimeric glycoprotein in order to relocalize it into lysosomes. Here, it was shown that, in cells transfected with plasmid that encodes glycoprotein with c-CD63 motif at the C-terminus, the chimeric protein was predominantly observed in lysosomes and at the cell membrane where the unmodified glycoprotein is localized in the endoplasmic reticulum and at the cell surface. We suppose that current modification of the glycoprotein may improve the immunogenicity of anti-rabies DNA vaccines due to more efficient antibody production.     

Newcastle disease virus-vectored rabies vaccine is safe, highly immunogenic, and provides long-lasting protection in dogs and cats

Effective, safe, and affordable rabies vaccines are still being sought. Newcastle disease virus (NDV), an avian paramyxovirus, has shown promise as a vaccine vector for mammals. Here, we generated a recombinant avirulent NDV La Sota strain expressing the rabies virus glycoprotein (RVG) and evaluated its potential to serve as a vaccine against rabies. The recombinant virus, rL-RVG, retained its high-growth property in chicken eggs, with titers of up to 10^9.8 50% egg infective doses (EID₅₀)/ml of allantoic fluid. RVG expression enabled rL-RVG to spread from cell to cell in a rabies virus-like manner, and RVG was incorporated on the surface of the rL-RVG viral particle. RVG incorporation did not alter the trypsin-dependent infectivity of the NDV vector in mammalian cells. rL-RVG and La Sota NDV showed similar levels of sensitivity to a neutralization antibody against NDV and similar levels of resistance to a neutralization antibody against rabies virus. Animal studies demonstrated that rL-RVG is safe in several species, including cats and dogs, when administered as multiple high doses of recombinant vaccine. Intramuscular vaccination with rL-RVG induced a substantial rabies virus neutralization antibody response and provided complete protection from challenge with circulating rabies virus strains. Most importantly, rL-RVG induced strong and long-lasting protective neutralization antibody responses to rabies virus in dogs and cats. A low vaccine dose of 10^8.3 EID₅₀ completely protected dogs from challenge with a circulating strain of rabies virus for more than a year. This is the first study to demonstrate that immunization with an NDV-vectored vaccine can induce long-lasting, systemic protective immunity against rabies.     

Sickness and recovery of dogs challenged with a street rabies virus after vaccination with a vaccinia virus recombinant expressing rabies virus N protein.

Dogs were vaccinated intradermally with vaccinia virus recombinants expressing the rabies virus glycoprotein (G protein) or nucleoprotein (N protein) or a combination of both proteins. The dogs vaccinated with either the G or G plus N proteins developed virus-neutralizing antibody titers, whereas those vaccinated with only the N protein did not. All dogs were then challenged with a lethal dose of a street rabies virus, which killed all control dogs. Dogs vaccinated with the G or G plus N proteins were protected. Five (71%) of seven dogs vaccinated with the N protein sickened, with incubation periods 3 to 7 days shorter than that of the control dogs; however, three (60%) of the five rabid dogs recovered without supportive treatment. Thus, five (71%) of seven vaccinated with the rabies N protein were protected against a street rabies challenge. Our data indicate that rabies virus N protein may be involved in reducing the incubation period in dogs primed with rabies virus N protein and then challenged with a street rabies virus and, of more importance, in subsequent sickness and recovery.     

Oral vaccination of skunks with raccoon poxvirus recombinants expressing the rabies glycoprotein or the nucleoprotein.

Twenty nine skunks (Mephitis mephitis) were vaccinated orally with raccoon poxvirus (RCN) recombinants: 10 with a recombinant expressing the rabies virus glycoprotein (RCNRG), 10 with RCNRG mixed with a recombinant expressing the rabies virus nucleoprotein (RCNRN) and nine with RCN alone. Rabies virus neutralizing antibodies were detected in six of the 20 skunks; five skunks (three given RCNRG, two given a mixture of recombinants) survived a rabies challenge that was lethal for nine skunks vaccinated with RCN alone.     

Vaccination of small Asian mongoose (Herpestes javanicus) against rabies

Oral vaccination of free-ranging wildlife is a promising technique in rabies control. The small Asian mongoose (Herpestes javanicus) is an important reservoir of rabies on several Caribbean islands, but no vaccines have been evaluated for this species. Captive mongooses were used to test the safety and efficacy of the commercially licensed vaccinia-rabies glycoprotein (V-RG) recombinant vaccine and a newly developed genetically engineered oral rabies virus vaccine (SPBNGA-S). In one study using V-RG, no vaccinated animals developed detectable rabies virus-neutralizing antibodies, and all but one died after experimental challenge with rabies virus. In contrast, all animals given SPBNGA-S demonstrated seroconversion within 7 to 14 days after vaccination and survived rabies virus challenge. On the basis of these preliminary results indicating the greater efficacy of SPBNGA-S vs. V-RG vaccine, additional investigations will be necessary to determine the optimal dose and duration of vaccination, as well as incorporation of the SPBNGA-S vaccine into edible bait.     

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.     

A recombinant human Fab expressed in Escherichia coli neutralizes rabies virus.

A recombinant human anti-rabies monoclonal antibody (MAb-57) Fab was prepared by cloning the heavy (Fd)- and light-chain domains into the same bacterial expression vector. To construct the recombinant Fab, mRNA was extracted from MAb-57-producing hybridoma cells, reverse transcribed, and then amplified by polymerase chain reaction (PCR) by using oligonucleotides specific for immunoglobulin heavy- and light-chain DNA sequences. PCR-amplified Fd-chain cDNA was fused, in frame, between a bacterial leader peptide (PelB) at the amino terminus and a 10-amino-acid peptide tag at the carboxy terminus. The PCR-amplified lambda-chain cDNA was also fused to the PelB leader peptide. The immunoglobulin Fab was then expressed as a dicistronic message in bacteria by using the isopropyl-beta-D-thiogalactopyranoside-inducible lactose promotor (lacZ). DNA sequencing was used to define the gamma-chain isotype (immunoglobulin G1) and VH (VHI) chain and VL (V lambda II) chain gene usage. The recombinant Fab (rFab57) specifically bound the rabies virus coat glycoprotein, while the Fd and lambda chains, when expressed individually, did not. The binding specificity of rFab57 was indistinguishable from that of the intact MAb in direct enzyme-linked immunosorbent assays; however, the dissociation constant of rFab57 for rabies virus protein G was approximately 1 log10 U lower than that of complete MAb-57 in competition enzyme-linked immunosorbent assays. A fluorescent-focus inhibition assay showed that bacterially expressed rFab was capable of neutralizing rabies virus strain CVS-11. We conclude that a human Fab expressed in bacteria maintains its specificity and biologic activity.