Human Rabies Vaccine Research Articles

A novel in vitro ELISA for estimation of glycoprotein content in human rabies vaccines

ABSTRACTIn vitro methods for quantification of immunodominant glycoprotein in the rabies vaccine formulations serve as good alternative to the cumbersome and variable mice potency assay as a batch release test for the vaccine. The present study presents the development of a sandwich ELISA with optimal concentrations of a high affinity recombinant diabody (D06) and a specific monoclonal antibody (M5B4) against rabies glycoprotein for its quantification in the vaccine formulations. The glycoprotein estimate correlated linearly (r2 = 0.8) to the in vivo potency estimate for the vaccine formulations. This ELISA promises a good forecast of the mice potency values and thereby can serve as a simple, yet effective batch release test for the rabies vaccines replacing the in vivo assay.

Replacement of in vivo human rabies vaccine potency testing by in vitro glycoprotein quantification using ELISA – Results of an international collaborative study

Three different ELISAs quantifying rabies glycoprotein were evaluated as in vitro alternatives to the National Institutes of Health (NIH) in vivo potency test for batch release of human rabies vaccines. The evaluation was carried out as an international collaborative study supported by the European Partnership for Alternatives to Animal Testing (EPAA). This pre-validation study, the results of which are presented in this paper, compared three different ELISA designs, assessing their within- and between-laboratory precision. One of the ELISA designs was proposed to the European Directorate for the Quality of Medicines & HealthCare (EDQM) and accepted for an international collaborative study under the umbrella of the Biological Standardisation Programme.

Expression of rabies virus glycoprotein in the methylotrophic yeast Pichia pastoris.

Rabies is a fatal disease that can be prevented by vaccination. Different approaches were investigated to develop novel human rabies vaccines with improved features compared to the current available vaccines, among them is the use of heterologous gene expression technology. Here, we describe the expression of the surface rabies virus glycoprotein (RABV-G), which is the major antigen responsible for the induction of protective immunity, in Pichia pastoris. Six transformants were selected according to their gene copy number as determined by real time qPCR. Upon induction by methanol, low level of RABV-G was secreted into the culture medium, around 60 ng/mL. To understand the effect of foreign gene dosage on cellular physiology of P. pastoris, transcriptional analysis of key genes involved in unfolded protein response (UPR) and endoplasmic reticulum associated degradation (ERAD) pathway was performed. Results showed that these pathways were highly activated; misfolded RABV-G was degraded in the cytosol via the ERAD mechanism. To study the functionality of the secreted RABV-G, in vitro competitive neutralizing assay was conducted. Data showed the secreted recombinant RABV-G had enabled a reduction of the neutralizing activity of human immune rabies serum, indicating that the secreted recombinant protein had reached its correct conformational form.

Current Situation of Rabies Prevention and Control in Developing Countries: Ethiopia Perspective

Rabies is a neglected zoonotic disease which kills up to 60,000 people a year, most of them in Africa and Asia. In the majority of developing countries, the number of patients receiving post exposure prophylaxis has steadily increased over time, particularly in urban areas due to dog related rabies. Studies conducted in sub-Saharan Africa show that most of the rabies cases in animals and humans are caused by canine rabies virus, mostly transmitted by domestic dogs and thus comprehensive and sustained dog vaccination is sufficient intervention in reduction and eventual elimination of human rabies in the region. In many developing countries, progress in preventing human rabies through control of the disease in the dog reservoir were slow due to technical, intersectoral, organizational and financial barriers. For developing countries like Ethiopia, strategy should be developed to prevent and control the disease. Sustainable resources for effective dog vaccination are likely to be available through the development of intersectoral financing schemes involving both medical and veterinary sectors. Prevention of animal rabies through dog vaccination, better public awareness, improved access to cost-effective and high-quality human rabies vaccines, and improved local capacity in rabies surveillance and diagnostics are essential for the elimination of human rabies. Generally, elimination of canine rabies is epidemiologically and practically feasible through mass vaccination of domestic dogs which is cost-effective approach to the prevention and elimination of human rabies deaths. The purpose of this paper is to show the burden of the disease and to give direction for effective prevention and control following developed countries experience.

Removing residual DNA from Vero-cell culture-derived human rabies vaccine by using nuclease

The clearance of host cell DNA is a critical indicator for Vero-cell culture-derived rabies vaccine. In this study, we evaluated the clearance of DNA in Vero-cell culture-derived rabies vaccine by purification process utilizing ultrafiltration, nuclease digestion, and gel filtration chromatography. The results showed that the bioprocess of using nuclease decreased residual DNA. Dot-blot hybridization analysis showed that the residual host cell DNA was <100 pg/ml in the final product. The residual nuclease in rabies vaccine was less than 0.1 ng/ml protein. The residual nuclease could not paly the biologically active role of digestion of DNA. Experiments of stability showed that the freeze-drying rabies virus vaccine was stable and titers were >5.0 IU/ml. Immunogenicity test and protection experiments indicated mice were greatly induced generation of neutralizing antibodies and invoked protective effects immunized with intraperitoneal injections of the rabies vaccine. These results demonstrated that the residual DNA was removed from virus particles and nuclease was removed by gel filtration chromatography. The date indicated that technology was an efficient method to produce rabies vaccine for human use by using nuclease.

In memoriam: Robert Emmons Kissling (1923–2013).

In memoriam: Robert Emmons Kissling (1923–2013).

The adaptation of a CTN-1 rabies virus strain to high-titered growth in chick embryo cells for vaccine development.

BackgroundRabies virus is the causative agent of rabies, a central nervous system disease that is almost invariably fatal. Currently vaccination is the most effective strategy for preventing rabies, and vaccines are most commonly produced from cultured cells. Although the vaccine strains employed in China include CTN, aG, PM and PV, there are no reports of strains that are adapted to primary chick embryo cells for use in human rabies prevention in China.ResultsRabies virus strain CTN-1 V was adapted to chick embryo cells by serial passage to obtain the CTNCEC25 strain. A virus growth curve demonstrated that the CTNCEC25 strain achieved high titers in chick embryo cells and was nonpathogenic to adult mice by intracerebral inoculation. A comparison of the structural protein genes of the CTNCEC25 strain and the CTN-1 V strain identified eight amino acid changes in the mature M, G and L proteins. The immunogenicity of the CTNCEC25 strain increased with the adaptation process in chick embryo cells and conferred high protective efficacy. The inactivated vaccine induced high antibody responses and provided full protection from an intramuscular challenge in adult mice.ConclusionsThis is the first description of a CTNCEC25 strain that was highly adapted to chick embryo cells, and both its in vitro and in vivo biological properties were characterized. Given the high immunogenicity and good propagation characteristics of the CTNCEC25 strain, it has excellent potential to be a candidate for development into a human rabies vaccine with high safety and quality characteristics for controlling rabies in China.

A relevant in vitro ELISA test in alternative to the in vivo NIH test for human rabies vaccine batch release

To assess the quality of vaccine batches before release, international regulation requires the control of potency of each lot of human rabies vaccines by the in vivo NIH challenge test. Meanwhile, the 3Rs strategy for animal testing encourages the replacement of the in vivo potency test by an in vitro assay. Consequently, since more than 10 years, an ELISA method has been implemented by ANSM in parallel to the NIH test for rabies vaccines lots. It consists in the evaluation of the glycoprotein content using a monoclonal antibody recognizing the trimeric native form of the glycoprotein. This ELISA method is able 1) to monitor the consistency of production with a similar profile than the NIH; 2) to detect a low quantity of glycoprotein in vaccines and 3) to agree with the manufacturer's NIH results by declaring a non compliant batch. This ELISA which characterizes the immunogenic form of the glycoprotein formulated in vaccines seems to be relevant to replace the NIH test and is a promising candidate to be standardized by a collaborative study.

Immunogenicity of a modified intradermal pre-exposure rabies vaccination schedule using a purified chick embryo cell vaccine: An observational study

In Australia, recommendations for pre-exposure rabies vaccination involve 3 doses of vaccines on days 0, 7, and 28 using either 1.0mL intramuscular (IM) vaccines, or 0.1mL intradermal (ID) vaccines. The use of IM vaccines is limited by their prohibitive cost, and ID vaccines by the recommendation for serology 2-3 weeks post-vaccination. A recent study reported the successful use of human diploid cell rabies vaccines (HDCV) with a modified ID schedule (2×0.1mL ID on days 0 and 7) - Travellers Rabies Intradermal 2 site (TRID2) - that was affordable, required less time to complete than standard ID schedules, and achieved a seroconversion rate of 94.5%. This study reports the immunogenicity of pre-exposure rabies vaccination using a purified chick embryo cell vaccine (PCECV) with the TRID2 schedule. Travellers aged 10-50 years were given the PCECV using the TRID2 schedule, and serology was performed at day 28. Fifty-four travellers were vaccinated, with a seroconversion rate of 94.4% at day 28. Seroconversion rates did not differ between age groups, but older travellers had lower antibody levels. This study supports the effectiveness of the TRID2 schedule, and found the schedule equally effective with HDCV or PCECV.

Reported Rabies Pre-exposure Immunization of Students at US Colleges of Veterinary Medicine

In 2008, the US experienced a disruption in human rabies vaccine supplies, leading public health authorities to prioritize vaccine release for post-exposure prophylaxis (PEP) and limit vaccine supplies for pre-exposure prophylaxis (PreEP) in high-risk groups. In 2008, the Association of American Veterinary Medical Colleges (AAVMC) surveyed its member institutions on rabies vaccination policies and practices. Senior administrators at Colleges of Veterinary Medicine (CVMs) and departments of veterinary science and comparative medicine were asked to identify the person most knowledgeable about their institution's student rabies vaccination program. Respondents were asked to describe their policies and procedures for administering PreEP to veterinary medical students and staff and to estimate the annual demand for student and staff PreEP vaccine. Twenty-one CVMs responded. Twenty (95%) reported requiring PreEP of veterinary medical students and 16 (80%) of those 20 required vaccination upon matriculation. An estimated 7,309 doses of vaccine were required for PreEP of an estimated 2,436 first-year US veterinary medical students. Seventy-two percent of respondents administered PreEP in August, September, and October, coinciding with the highest public demand for PEP. CVMs should consider altering the timing of rabies vaccine administration to veterinary medical students and staff to other months, thereby helping to ensure that PEP rabies vaccine will be available to people with validated rabies exposures and to ensure that supplies will be available for PreEP of students and staff. AAVMC may wish to identify and support a point of coordination to facilitate the purchase and distribution of human rabies vaccine among its US member CVMs.

Reinvestigating the role of IgM in rabies virus postexposure vaccination.

B cells secreting IgG antibodies, but not IgM, are thought to be solely responsible for vaccine-induced protection against rabies virus (RABV) infections in postexposure settings. In this report, we reinvestigated the potential for IgM to mediate protection in a mouse model of RABV vaccination. Immunocompetent mice immunized with an experimental live replication-deficient RABV-based vaccine produced virus neutralizing antibodies (VNAs) within 3 days of vaccination. However, mice unable to produce soluble IgM (sIgM(-/-)) did not produce VNAs until 7 days postimmunization. Furthermore, sIgM(-/-) mice were not protected against RABV infection when challenged 3 days postimmunization, while all wild-type mice survived challenge. Consistent with the lack of protection against pathogenic RABV challenge, approximately 50- to 100-fold higher viral loads of challenge virus were detected in the muscle, spinal cord, and brain of immunized sIgM(-/-) mice compared to control mice. In addition, IgG antibody titers in vaccinated wild-type and sIgM(-/-) mice were similar at all time points postimmunization, suggesting that protection against RABV challenge is due to the direct effects of IgM and not the influence of IgM on the development of effective IgG antibody titers. In all, early vaccine-induced IgM can limit dissemination of pathogenic RABV to the central nervous system and mediate protection against pathogenic RABV challenge. Considering the importance for the rapid induction of VNAs to protect against RABV infections in postexposure prophylaxis settings, these findings may help guide the development of a single-dose human rabies vaccine.

Molecular characterization of China human rabies vaccine strains

To understand the molecular characteristics of China human rabies vaccine strains, we report the full-length genome of the aG strain and present a comprehensive analysis of this strain and almost all available lyssavirus genomes (58 strains) from GenBank (as of Jan 6, 2011). It is generally considered that the G protein plays a predominant role in determining the pathogenicity of the virus, to this end we predicted the tertiary structure of the G protein of aG strain, CTN181 strain and wild type strain HN10 based on the crystal structure of Vesicular stomatitis virus (VSV) G. The predicted RABV G structure has a similar topology to VSV G and the ectodomain can be divided into 4 distinct domains DI - DIV. By mapping the characterized mutations to this structure between China vaccine strains and their close street strains, we speculate that the G303(P-H) mutations of CTN181 and HN10 causing DII 3D change may be associated with the II attenuated virulence in both strains. Specifically, the two signature mutations (G165P and G231P) in the aG strain are within ßsheets, suggesting that both sites are of structural importance.

Assessment of inactivated human rabies vaccines: Biochemical characterization and genetic identification of virus strains

The World Health Organization (WHO) recommends the periodic evaluation of the purity of the cell lines used in the production of rabies vaccines, as well as the antigenic identity of the virus strains. Here, we analyzed seventeen marketed inactivated human rabies virus vaccines for viral and non-viral proteins by SDS-PAGE and Coomassie/silver staining. Mass spectrometric analysis of an abundant 60–70kDa signal indicated that in most vaccines serum albumin of human origin (HSA) was the major component. Quantification of HSA in the vaccines revealed a mean concentration of 22mg HSA/dose in all tested PVRV (purified vero cell rabies vaccine), HDCV (human diploid cell rabies vaccine) and PHK (primary hamster kidney) vaccines. In contrast, 1000-fold lower HSA levels and no HSA were detected in PCECV (purified chick embryo cell-culture vaccine) and PDEV (duck embryo rabies vaccine), respectively. Western blot analyses further confirmed a high bias in the HSA content, whereas the virus protein levels were rather similar in all tested vaccines. In addition, the vaccine viruses were sequenced within the N- and G-genes to identify the strain. In the majority of sequenced vaccines, the declared vaccine strain was confirmed. However, some discrepancies in the genetic identification were observed, supporting WHO's recommendation for the molecular characterization of vaccine seed strains. This research highlights the variation in purity found between different human rabies virus vaccines, and suggests that further research is needed to establish the impact non-active components have on the potency of such vaccines.

Molecular characterization of China rabies virus vaccine strain

BackgroundRabies virus (RV), the agent of rabies, can cause a severe encephalomyelitis in several species of mammals, including humans. As a human rabies vaccine strain employed in China, the genetic knowledge of the aG strain has not been fully studied. The main goal of the present study is to amplify the whole genome of aG strain, and genetic relationships between other vaccine strains and wild strains were analyzed.ResultsThe entire genome of human rabies virus vaccine strain aG employed in China was sequenced; this is the second rabies virus vaccine strain from China to be fully characterized. The overall organization and the length of the genome were similar to that of other lyssaviruses. The length of aG strain was 11925nt, comprising a leader sequence of 58nt, nucleoprotein (N) gene of 1353nt, phosphoprotein (P) gene of 894 nt, matrix protein (M) gene of 609nt, glycoprotein (G) gene of 1575nt, RNA-dependent RNA polymerase (RdRp,L) gene of 6384nt, and a trailer region of 70 nt. There was TGAAAAAAA (TGA7) consensus sequence in the end of each gene, except AGA7 at the end of G gene. There was AACAYYYCT consensus start signal at the beginning of each gene.ConclusionsIn this report, we analyzed the full genome of China human rabies vaccine strain aG. Our studies indicated that the genome of aG retained the basic characteristics of RV. At gene level, N was the most conserved among the five coding genes, indicating this gene is the most appropriate for quantitative genotype definition. The phylogenetic analysis of the N indicated the aG strain clustered most closely with Japanese and Russian rabies vaccine strains, suggesting that they may share the same ancestor; also, the aG strain did not share high homology with wild strains isolated from China, making it may not be the best vaccine strain, more research is needed to elucidate the genetic relationship among the RV circulating in China.

From brain passage to cell adaptation: the road of human rabies vaccine development

A major challenge for global rabies prevention and control is the lack of sufficient and affordable high quality vaccines. Such candidates should be pure, potent, safe, effective and economical to produce, with broad cross-reactivity against viral variants of public health and veterinary importance. The history of licensed human vaccines reviewed herein demonstrates clearly how the field has evolved to the current state of more passive development and postexposure management. Modern cell culture techniques provide adequate viral substrates for production of representative verified virus seeds. In contrast to outdated nervous tissue-based rabies vaccines, once a suitable substrate is identified, production of high titer virus results in a major qualitative and quantitative difference. Given the current scenario of only inactivated vaccines for humans, highly cell-adapted and stable, attenuated rabies viruses are ideal candidates for consideration to meet the need for seed viruses in the future.

El proceso de creación del Instituto Pasteur (1886–1888) según la prensa española de la época

With the discovery of the human rabies vaccine, Louis Pasteur (1822–1895) introduced a new perspective, inaugurating the second phase of vaccinology. The impact of his discovery was amplified by the press, which represented him as a universal hero and man of science. This study explores how the initiative to create an institute in Paris bearing Pasteur's name was represented in the Spanish press during the period 1886–1888. The institute was initially dedicated to the study and treatment of rabies and was later widened to include other diseases and their vaccinations.

Development of recombinant rabies viruses vectors with Gaussia luciferase reporter based on Chinese vaccine strain CTN181

The recombinant rabies virus (RV) vectors encoding the secreted gene marker Gaussia luciferase (Gluc) were generated based on Chinese vaccine strain CTN181. Vectors included replication competent CTN-Gluc, CTN/GQ333R-Gluc, in which the amino acid in position 333 of glycoprotein was mutated from glutamine (Q) to arginine (R), and replication constrained CTNΔG-Gluc, in which the glycoprotein encoding gene (G) was deleted. The growth of recombinant RVs in transfected cells was confirmed through biochemical assays of Gluc activities. Gluc expression in recombinant CTNΔG-Gluc virus was highest while that in CTN/GQ333R-Gluc virus was lowest. The optimal time to harvest recombinant RVs was determined and the function of pathogenic and nonpathogenic rabies glycoprotein in virus recovery was examined. The addition of glycoprotein was slightly beneficial for virus recovery and the titer of rescued virus was lowered even when the amino acid in G333 position of glycoprotein was mutated from nonpathogenic Gln to pathogenic Arg. Conclusions: Viral vectors based on a human rabies vaccine strain CTN181 were successful. Gluc was useful as an in vitro gene marker for monitoring the growth of recombinant RVs iteratively in cell culture.

Live attenuated rabies virus co-infected with street rabies virus protects animals against rabies

While current rabies post-exposure prophylaxis (PEP) is highly effective, it is costly and the vaccination regimen is complicated, requiring both inactivated vaccines and immunoglobulins. A one-dose rabies vaccine for human PEP remains a long-term goal. Here, we describe development of a highly attenuated rabies virus ERAg3m, with a mutation in the glycoprotein (G) gene and a switch of the G gene with the matrix protein gene in the viral genome. After a one-dose intramuscular vaccination, the ERAg3m virus protected 100% of mice and hamsters from lethal challenge. In co-infections, using a lethal dose of street rabies virus mixed with ERAg3m, 100% of hamsters and 90% of mice survived and were protected against subsequent infection. A mock co-infection, using inactivated commercial human rabies vaccine and a lethal dose of street rabies virus, protected 100% and 40% of hamsters and mice, respectively. In co-infections, when vaccine was administrated in the left leg and challenge virus in the right leg, the ERAg3m virus protected 40% of mice, while the inactivated vaccine showed no protection. Therefore, live attenuated rabies virus when given pre-exposure or co-infected with street rabies virus, is capable of preventing rabies in two different animal models. Overall, this highly attenuated live rabies virus offered better protection than the inactivated vaccine.

Efficacy of RTS,S/AS01E malaria vaccine and exploratory analysis on anti-circumsporozoite antibody titres and protection in children aged 5–17 months in Kenya and Tanzania: a randomised controlled trial

SummaryBackgroundRTS,S/AS01E is the lead candidate malaria vaccine. We recently showed efficacy against clinical falciparum malaria in 5–17 month old children, during an average of 8 months follow-up. We aimed to assess the efficacy of RTS,S/AS01E during 15 months of follow-up.MethodsBetween March, 2007, and October, 2008, we enrolled healthy children aged 5–17 months in Kilifi, Kenya, and Korogwe, Tanzania. Computer-generated block randomisation was used to randomly assign participants (1:1) to receive three doses (at month 0, 1, and 2) of either RTS,S/AS01E or human diploid-cell rabies vaccine. The primary endpoint was time to first clinical malaria episode, defined as the presence of fever (temperature ≥37·5°C) and a Plasmodium falciparum density of 2500/μL or more. Follow-up was 12 months for children from Korogwe and 15 months for children from Kilifi. Primary analysis was per protocol. In a post-hoc modelling analysis we characterised the associations between anti-circumsporozoite antibodies and protection against clinical malaria episodes. This study is registered with ClinicalTrials.gov, number NCT00380393.Findings894 children were assigned, 447 in each treatment group. In the per-protocol analysis, 82 of 415 children in the RTS,S/AS01E group and 125 of 420 in the rabies vaccine group had first or only clinical malaria episode by 12 months, vaccine efficacy 39·2% (95% CI 19·5–54·1, p=0·0005). At 15 months follow-up, 58 of 209 children in the RTS,S/AS01E group and 85 of 206 in the rabies vaccine group had first or only clinical malaria episode, vaccine efficacy 45·8% (24·1–61·3, p=0·0004). At 12 months after the third dose, anti-circumsporozoite antibody titre data were available for 390 children in the RTS,S/AS01E group and 391 in the rabies group. A mean of 15 months (range 12–18 months) data were available for 172 children in the RTS,S/AS01E group and 155 in the rabies group. These titres at 1 month after the third dose were not associated with protection, but titres at 6·5 months were. The level of protection increased abruptly over a narrow range of antibody concentrations. The most common adverse events were pneumonia, febrile convulsion, gastroenteritis, and P falciparum malaria.InterpretationRTS,S/AS01E confers sustained efficacy for at least 15 months and shows promise as a potential public health intervention against childhood malaria in malaria endemic countries.FundingPATH Malaria Vaccine Initiative (MVI), GlaxoSmithKline.

Experimental rabies vaccines for humans

Rabies remains a global public health threat that kills more than 55,000 people per year. Rabies disproportionately affects children and, therefore, is ranked the seventh most important infectious disease due to years lost. Prevention of human rabies is accomplished by controlling rabies in domestic and wild animals, including the use of vaccination programs. The usefulness of human rabies vaccines is hampered by high cost, complicated vaccination regimens and lack of compliance, especially in areas of Africa and Asia where human rabies infections are endemic. A single-dose vaccine would greatly benefit efforts to combat this global health threat. However, a single-dose vaccine based on current inactivated vaccines does not appear feasible and other approaches are needed. Technology has advanced since modern human rabies vaccines were developed over 40 years ago. In addition, our understanding of immunological principles that influence the outcome of vaccination has increased. This article describes the current status of inactivated rabies virus vaccines and recent developments arising from the use of reverse genetics technologies designed to develop replication-deficient or single-cycle live rabies virus-based vectors for use as a single-dose rabies vaccine for humans.