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1.  Using Serology to Assist with Complicated Post-Exposure Prophylaxis for Rabies and Australian Bat Lyssavirus 
Background
Australia uses a protocol combining human rabies immunoglobulin (HRIG) and rabies vaccine for post-exposure prophylaxis (PEP) of rabies and Australian bat lyssavirus (ABLV), with the aim of achieving an antibody titre of ≥0.5 IU/ml, as per World Health Organization (WHO) guidelines, as soon as possible.
Methodology/Principal Findings
We present the course of PEP administration and serological testing for four men with complex requirements. Following dog bites in Thailand, two men (62 years old, 25 years old) received no HRIG and had delayed vaccine courses: 23 days between dose two and three, and 18 days between dose one and two, respectively. Both seroconverted following dose four. Another 62-year-old male, who was HIV-positive (normal CD4 count), also suffered a dog bite and had delayed care receiving IM rabies vaccine on days six and nine in Thailand. Back in Australia, he received three single and one double dose IM vaccines followed by another double dose of vaccine, delivered intradermally and subcutaneously, before seroconverting. A 23-year-old male with a history of allergies received simultaneous HRIG and vaccine following potential ABLV exposure, and developed rash, facial oedema and throat tingling, which was treated with a parenteral antihistamine and tapering dose of steroids. Serology showed he seroconverted following dose four.
Conclusions/Significance
These cases show that PEP can be complicated by exposures in tourist settings where reliable prophylaxis may not be available, where treatment is delayed or deviates from World Health Organization recommendations. Due to the potentially short incubation time of rabies/ABLV, timely prophylaxis after a potential exposure is needed to ensure a prompt and adequate immune response, particularly in patients who are immune-suppressed or who have not received HRIG. Serology should be used to confirm an adequate response to PEP when treatment is delayed or where a concurrent immunosuppressing medical condition or therapy exists.
Author Summary
In Australia, the administration of rabies post-exposure prophylaxis (PEP) occurs for potentially exposed returned travellers from endemic regions or for potential local exposure to Australian Bat Lyssavirus. For Australian tourists, delays in commencing PEP or not receiving HRIG or all recommended doses of vaccine are common. We report a case series where serology provided information in four patients with delayed, incomplete, or complicated PEP treatment. Three of these patients reported a dog bite in Thailand and the fourth was scratched by a bat and had bat urine enter his eye in Australia. Management was complicated by lack of HRIG administration, delays in the recommended timeframe for receipt of vaccine doses, and immunosuppression caused by co-administration of steroids and by HIV infection with a normal CD4 count. All patients seroconverted but this was delayed in some cases, and in the HIV-positive subject required a double dose of vaccine delivered intradermally and subcutaneously. In complex or non-standard PEP delivery, including delayed treatment and immunosuppression due to steroid treatment, HIV or another immunosuppressing medical condition, serology can be used to guide further treatment and should be used to confirm seroconversion.
doi:10.1371/journal.pntd.0002066
PMCID: PMC3584984  PMID: 23469301
2.  A Simplified 4-Site Economical Intradermal Post-Exposure Rabies Vaccine Regimen: A Randomised Controlled Comparison with Standard Methods 
Background
The need for economical rabies post-exposure prophylaxis (PEP) is increasing in developing countries. Implementation of the two currently approved economical intradermal (ID) vaccine regimens is restricted due to confusion over different vaccines, regimens and dosages, lack of confidence in intradermal technique, and pharmaceutical regulations. We therefore compared a simplified 4-site economical PEP regimen with standard methods.
Methods
Two hundred and fifty-four volunteers were randomly allocated to a single blind controlled trial. Each received purified vero cell rabies vaccine by one of four PEP regimens: the currently accepted 2-site ID; the 8-site regimen using 0.05 ml per ID site; a new 4-site ID regimen (on day 0, approximately 0.1 ml at 4 ID sites, using the whole 0.5 ml ampoule of vaccine; on day 7, 0.1 ml ID at 2 sites and at one site on days 28 and 90); or the standard 5-dose intramuscular regimen. All ID regimens required the same total amount of vaccine, 60% less than the intramuscular method. Neutralising antibody responses were measured five times over a year in 229 people, for whom complete data were available.
Findings
All ID regimens showed similar immunogenicity. The intramuscular regimen gave the lowest geometric mean antibody titres. Using the rapid fluorescent focus inhibition test, some sera had unexpectedly high antibody levels that were not attributable to previous vaccination. The results were confirmed using the fluorescent antibody virus neutralisation method.
Conclusions
This 4-site PEP regimen proved as immunogenic as current regimens, and has the advantages of requiring fewer clinic visits, being more practicable, and having a wider margin of safety, especially in inexperienced hands, than the 2-site regimen. It is more convenient than the 8-site method, and can be used economically with vaccines formulated in 1.0 or 0.5 ml ampoules. The 4-site regimen now meets all requirements of immunogenicity for PEP and can be introduced without further studies.
Trial Registration
Controlled-Trials.com ISRCTN 30087513
Author Summary
All human deaths from rabies result from failure to give adequate prophylaxis. After a rabid animal bite, immediate wound cleaning, rabies vaccine and immunoglobulin injections effectively prevent fatal infection. Immunoglobulin is very rarely available in developing countries, where prevention relies on efficacious vaccine. WHO approved vaccines are prohibitively expensive, but 2 economical regimens (injecting small amounts of vaccine intradermally, into the skin, at 2 or 8 sites on the first day of the course) have been used for many years in a few places. Practical or perceived difficulties have restricted widespread uptake of economical methods. These could largely be overcome by introducing a new, simpler regimen, involving 4 site injections on the first day. We vaccinated volunteers to compare the antibody levels induced by the 4-site intradermal regimen with those induced by the current 2-site and 8-site regimens and the “gold standard” intramuscular regimen favoured internationally. All the economical intradermal regimens were at least as immunogenic as the intramuscular method. The results provide sufficient evidence that the 4-site regimen meets the criteria necessary for its recommendation for use wherever the cost of vaccine is prohibitive and especially where 2 or more patients are treated on the same day.
doi:10.1371/journal.pntd.0000224
PMCID: PMC2292256  PMID: 18431444
3.  Rabies trend in China (1990–2007) and post-exposure prophylaxis in the Guangdong province 
Background
Rabies is a major public-health problem in developing countries such as China. Although the recent re-emergence of human rabies in China was noted in several epidemiological studies, little attention was paid to the reasons behind this phenomenon paralleling the findings of the previous reports. The purpose of this study is thus first to characterize the current trends of human rabies in China from 1990 to 2007, and then to define better recommendations for improving the post-exposure prophylaxis (PEP) schedules delivered to rabies patients.
Methods
The most updated epidemiological data for 22527 human rabies cases from January 1990 to July 2007, retrieved from the surveillance database of reportable diseases managed by the Ministry of Health of China, were analysed. To investigate the efficiency for the post-exposure treatment of rabies, the details of 244 rabies patients, including their anti-rabies treatment of injuries or related incidents, were ascertained in Guangdong provincial jurisdiction. The risk factors to which the patients were predisposed or the regimens given to 80 patients who received any type of PEP were analysed to identify the reasons for the PEP failures.
Results
The results from analysis of the large number of human rabies cases showed that rabies in China was largely under control during the period 1990–1996. However, there has been a large jump in the number of reported rabies cases since 2001 up to a new peak (with an incidence rate of 0.20 per 100000 people) that was reached in 2004, and where the level has remained until present. Then, we analysed the PEP in 244 rabies cases collected in the Guangdong province in 2003 and 2004, and found that 67.2% of the patients did not seek medical services or did not receive any PEP. Further analysis of PEP for the 80 rabies patients who received any type of PEP indicated that almost all of the patients did not receive proper or timely treatment on the wounds or post-exposure vaccination or rabies immunoglobulins.
Conclusion
While the issue of under-reporting of rabies in previous years may well be a factor in the apparent upwards trend of human rabies in recent years, the analysis of PEP in the Guangdong province provides evidence that suggests that the failure to receive PEP was a major factor in the number of human cases in China. Thus, the data underline the need for greatly improved availability and timely application of high-quality anti-rabies biologicals, both vaccines and immunoglobulins, in the treatment of human bite victims. Controlling dog rabies through pet vaccination schemes may also play a huge role in reducing the rate of human exposure. Education of the public, health care staff and veterinarians will also help to change the current situation.
doi:10.1186/1471-2334-8-113
PMCID: PMC2532688  PMID: 18717989
4.  Investigating the Role for IL-21 in Rabies Virus Vaccine-induced Immunity 
Over two-thirds of the world's population lives in regions where rabies is endemic, resulting in over 15 million people receiving multi-dose post-exposure prophylaxis (PEP) and over 55,000 deaths per year globally. A major goal in rabies virus (RABV) research is to develop a single-dose PEP that would simplify vaccination protocols, reduce costs associated with RABV prevention, and save lives. Protection against RABV infections requires virus neutralizing antibodies; however, factors influencing the development of protective RABV-specific B cell responses remain to be elucidated. Here we used a mouse model of IL-21 receptor-deficiency (IL-21R−/−) to characterize the role for IL-21 in RABV vaccine-induced immunity. IL-21R−/− mice immunized with a low dose of a live recombinant RABV-based vaccine (rRABV) produced only low levels of primary or secondary anti-RABV antibody response while wild-type mice developed potent anti-RABV antibodies. Furthermore, IL-21R−/− mice immunized with low-dose rRABV were only minimally protected against pathogenic RABV challenge, while all wild-type mice survived challenge, indicating that IL-21R signaling is required for antibody production in response to low-dose RABV-based vaccination. IL-21R−/− mice immunized with a higher dose of vaccine produced suboptimal anti-RABV primary antibody responses, but showed potent secondary antibodies and protection similar to wild-type mice upon challenge with pathogenic RABV, indicating that IL-21 is dispensable for secondary antibody responses to live RABV-based vaccines when a primary response develops. Furthermore, we show that IL-21 is dispensable for the generation of Tfh cells and memory B cells in the draining lymph nodes of immunized mice but is required for the detection of optimal GC B cells or plasma cells in the lymph node or bone marrow, respectively, in a vaccine dose-dependent manner. Collectively, our preliminary data show that IL-21 is critical for the development of optimal vaccine-induced primary but not secondary antibody responses against RABV infections.
Author Summary
Over two-thirds of the world's population lives in regions where rabies is endemic, resulting in over 15 million people receiving post-exposure treatment. A person, disproportionately a child, dies of rabies every 20 minutes and the cost of rabies prevention exceeds $1 billion US dollars per year. The development of a single-dose human rabies vaccine would greatly reduce the burden of rabies globally by lowering the cost associated with rabies vaccination and saving lives. Understanding how B cells develop to produce protective virus neutralizing antibodies would greatly help to achieve the goal of developing a single-dose vaccine. In this report, we show that IL-21 is critical for the induction of primary vaccine-induced anti-RABV G antibody titers and that the effects of IL-21 are highly dependent on the dose of vaccine administered. In our model of rabies immunogenicity and protection, the lack of IL-21 receptor influenced the detection of B cells in germinal centers in lymph nodes or of plasma cells in bone marrow after immunization with low or high doses of vaccine, respectively. Overall, these preliminary results indicate that IL-21 has the potential to influence B cell development and functions in the context of rabies vaccine-induced immunity and protection.
doi:10.1371/journal.pntd.0002129
PMCID: PMC3597479  PMID: 23516660
5.  Improvement Control System of Rabies in Ukraine 
Objective
The purpose of the research was to find out the reasons of rabies antropurgisation in Ukraine.
Introduction
In Ukraine in spite of considerable financial expenses on oral immunization of foxes and parenteral immunization of dogs and cats, it is not succeeded to reach considerable results in the fight with rabies. Unfortunately there was a negative tendency to increasing a part of dogs and cats in the structure of rabies disease which are the main source of rabies in people.
Methods
Analysis of 228 anamnesis data of rabies infected dogs during 2008–2012. Research of 234 samples of the blood serum from dogs on existence of antibodies to the rabies virus by the ELISA method.
Results
Analysis of animal morbidity on rabies in Ukraine in period of 2006–2011 found out the changes of structure of morbidity in animal species that means decreasing a part of wild animals (from 49,0 % in 2006 to 38,7 % in 2011) and increasing a part of dogs (from 18,3 % in 2006 to 23,2 % in 2011) and cats (from 19,8 % in 2006 to 25,0 % in 2011) in the general amount of animals which perished from rabies (Fig. 1).
A lot of Ukrainian scientists and doctors of veterinary medicine consider that the main reason of spreading the rabies is a great number of homeless animals which factually are the reservoirs of infection in towns and villages.
However, in our opinion spreading of rabies shows the insufficient level of measures of control of rabies among home animals. It was confirmed with conducted analysis that only 26 (12,9 %) dogs were stray, others 202 (87,1 %) had owners, but didn’t get necessary protective rabies vaccination.
According to Ukrainian instruction “Preventive measures against rabies of animals”, all the dogs must be vaccinated against rabies, but it actually appears it is quite not so.
At research of 234 samples of the blood serum of dogs on existence of antibodies to the rabies virus it was determined that the level of population immunity in dogs is 36,6 % in Ukrainian towns, but the protective level of antibodies was found in 9,1 % of village dogs. We consider that the main reason of this is imperfection of the Ukrainian legislation in the questions of responsibility of proprietors of animals.
For breaking of epizootic chain it is necessary the percent of vaccinated animals (in this case dogs) to be on a high level – more than 50%. Nowadays for reaching epidemic welfare in rabies it is important within the framework of the registered epidemic incidents dogs to be brought over to the ecocycles of infection sporadically without taking part in circulation of exciter, stay their biological deadlock and have low epidemic potential.
Conclusions
The conducted analysis expressly demonstrates that at present problems the eradication of rabies in Ukraine is impossible considering the low level of dogs’ protection from rabies. Obtained results were sent to the State committee of veterinary medicine of Ukraine and will be the argument for enhancement of control after conducting rabies vaccination of dogs.
Rabies cases in the years 2006–2011.
PMCID: PMC3692804
population rabies immunity; rabies vaccination; stray dogs
6.  The Production of Antibody by Invading B Cells Is Required for the Clearance of Rabies Virus from the Central Nervous System 
Background
The pathogenesis of rabies is associated with the inability to deliver immune effectors across the blood-brain barrier and to clear virulent rabies virus from CNS tissues. However, the mechanisms that facilitate immune effector entry into CNS tissues are induced by infection with attenuated rabies virus.
Methodology/Principal Findings
Infection of normal mice with attenuated rabies virus but not immunization with killed virus can promote the clearance of pathogenic rabies virus from the CNS. T cell activity in B cell–deficient mice can control the replication of attenuated virus in the CNS, but viral mRNA persists. Low levels of passively administered rabies virus–neutralizing antibody reach infected cells in the cerebellum of B cell–deficient mice but are not sufficient to mediate virus clearance. Production of rabies virus-specific antibody by B cells invading CNS tissues is required for this process, and a substantial proportion of the B cells that accumulate in the CNS of mice infected with attenuated rabies virus produce virus-specific antibodies.
Conclusions/Significance
The mechanisms required for immune effectors to enter rabies virus-infected tissues are induced by infection with attenuated rabies virus but not by infection with pathogenic rabies viruses or immunization with killed virus. T cell activities can inhibit rabies virus replication, but the production of rabies virus–specific antibodies by infiltrating B cells, as opposed to the leakage of circulating antibody across the BBB, is critical to elimination of the virus. These findings suggest that a pathogenic rabies virus infection may be treatable after the virus has reached the CNS tissues, providing that the appropriate immune effectors can be targeted to the infected tissues.
Author Summary
Every year over 50,000 people die from rabies worldwide, primarily due to the poor availability of rabies vaccine in developing countries. However, even when vaccines are available, human deaths from rabies occur if exposure to the causative virus is not recognized and vaccination is not sought in time. This is because rabies virus immunity induced by the natural infection or current vaccines is generally not effective at removing disease-causing rabies virus from brain tissues. Our studies provide insight into why this is the case and how vaccination can be changed so that the immune response can clear the virus from brain tissues. We show that the type of immune response induced by a live-attenuated rabies virus vaccine may be the key. In animal models, live-attenuated rabies virus vaccines are effective at delivering the immune cells capable of clearing the virus into CNS tissues and promote recovery from a rabies virus infection that has spread to the brain while conventional vaccines based on killed rabies virus do not. The production of rabies-specific antibody by B cells that invade the CNS tissues is important for complete elimination of the virus. We hypothesize that similar mechanisms may promote rabies virus clearance from individuals who are diagnosed after the virus has reached, but not extensively spread, through the CNS.
doi:10.1371/journal.pntd.0000535
PMCID: PMC2754506  PMID: 19806203
7.  Dog bites in Bosnia. 
BACKGROUND: Rabies is a zoonosis that remains endemic in most parts of the world. Primary care physicians are in the first line of defence against the disease. An increasing number of British practitioners and medical students are being exposed to the dangers of rabies through humanitarian work on overseas attachments. Rabies is enzootic throughout Bosnia-Herzegovina and presents a hazard to the multinational troops currently deployed there. AIM: To describe the British Army's experience of animal bites and rabies prevention in Bosnia during the first six months of its current peace enforcement mission, and to make general recommendations on the good management of any rabies hazard at primary care level and under field conditions. METHODS: Routine data from the Army's epidemiological database (ARRC 97) were reviewed, and theatre issues of rabies vaccine and immune globulin were used as a proxy measure for administered post-exposure prophylaxis. RESULTS: A total of 62 animal bites were reported in British troops between December 1995 and May 1996, of which 28 were unprovoked bites and resulted in the administration of a course of rabies vaccine. Ten of these were severe bites and rabies immune globulin (RIG) was administered in addition. The total cost of rabies post-exposure prophylaxis was US$6914.00. CONCLUSION: The prevention of rabies has major human and resource implications, and primary care staff involved in rabies post-exposure management need to be well supported in their clinical decision-making. Rabies protocols should be clear and unambiguous. The effective medical surveillance of military or humanitarian missions in rabies-enzootic areas must include the prompt reporting of animal bites. The predeployment training of medical teams should include an up-to-date assessment of the local rabies threat.
PMCID: PMC1313054  PMID: 9281871
8.  Achieving Population-Level Immunity to Rabies in Free-Roaming Dogs in Africa and Asia 
Canine rabies can be effectively controlled by vaccination with readily available, high-quality vaccines. These vaccines should provide protection from challenge in healthy dogs, for the claimed period, for duration of immunity, which is often two or three years. It has been suggested that, in free-roaming dog populations where rabies is endemic, vaccine-induced protection may be compromised by immuno-suppression through malnutrition, infection and other stressors. This may reduce the proportion of dogs that seroconvert to the vaccine during vaccination campaigns and the duration of immunity of those dogs that seroconvert. Vaccination coverage may also be limited through insufficient vaccine delivery during vaccination campaigns and the loss of vaccinated individuals from populations through demographic processes. This is the first longitudinal study to evaluate temporal variations in rabies vaccine-induced serological responses, and factors associated with these variations, at the individual level in previously unvaccinated free-roaming dog populations. Individual-level serological and health-based data were collected from three cohorts of dogs in regions where rabies is endemic, one in South Africa and two in Indonesia. We found that the vast majority of dogs seroconverted to the vaccine; however, there was considerable variation in titres, partly attributable to illness and lactation at the time of vaccination. Furthermore, >70% of the dogs were vaccinated through community engagement and door-to-door vaccine delivery, even in Indonesia where the majority of the dogs needed to be caught by net on successive occasions for repeat blood sampling and vaccination. This demonstrates the feasibility of achieving population-level immunity in free-roaming dog populations in rabies-endemic regions. However, attrition of immune individuals through demographic processes and waning immunity necessitates repeat vaccination of populations within at least two years to ensure communities are protected from rabies. These findings support annual mass vaccination campaigns as the most effective means to control canine rabies.
Author Summary
Canine-mediated rabies is a horrific disease that claims tens of thousands of human lives every year, particularly in Asia and Africa. The disease can be effectively controlled through mass vaccination of dogs with high-quality vaccines; however, questions remain over the effectiveness of vaccination where the health status of free-roaming dogs may be compromised and the life expectancy and access to these dogs may be limited. This study evaluated rabies-vaccine induced immune responses and vaccine delivery in previously unvaccinated, free-roaming dog populations in two rabies endemic regions in Asia and Africa, to better understand the effectiveness of vaccination campaigns. We found that the majority of dogs seroconverted to the vaccine regardless of health status. Excellent vaccination coverage was achieved through community engagement and door-to-door vaccine delivery, even where the majority of the dogs needed to be caught by net for vaccination. However, attrition of immune individuals through demographic processes and waning immunity reinforces the importance of frequent and regular vaccination campaigns to ensure effective vaccination coverage is maintained.
doi:10.1371/journal.pntd.0003160
PMCID: PMC4230884  PMID: 25393023
9.  Evaluation of a one week intradermal regimen for rabies post-exposure prophylaxis: Results of a randomized, open label, active-controlled trial in healthy adult volunteers in India 
Human Vaccines & Immunotherapeutics  2012;8(8):1077-1081.
The currently recommended intradermal regimen for post-exposure prophylaxis spreads over a month period which many times lead to low compliance from the patients. There is a need to introduce and evaluate short course regimens to overcome this problem. This study was conducted to evaluate the immunogenicity and safety of a “new one week intradermal regimen” for rabies post-exposure prophylaxis. A total of 80 healthy adult volunteers were enrolled and allocated randomly either to purified chick embryo cell (PCECV) rabies vaccine or purified verocell rabies vaccine (PVRV), 40 in each group. Each subject received intradermally one of the vaccines, using the one week regimen (4–4-4). Blood samples were collected on Days 0, 7, 14, 28,180 and 365 for estimation of rabies virus neutralizing antibody (RVNA) concentration. The sera samples were analyzed by rapid fluorescent focus inhibition test (RFFIT). All subjects in both the groups had adequate RVNA concentration of ≥ 0.5 IU/mL from day 14 to till day 180 and the difference of geometric mean concentrations between the two groups was not significant (p > 0.606). Further to assess the immunological memory produced by this new regimen, a “single visit four site” intradermal booster vaccination was given to those who did not have adequate RVNA concentration on day 365. This resulted in a quick and enhanced RVNA concentration in these subjects thus denoting a successful anamnestic response. The incidence of adverse events was 8.3% in PCECV group and 1.6% in PVRV group (p = 0.001) and the regimen was well tolerated without any dropouts. In conclusion, the new “one week intradermal regimen” is immunogenic and safe for rabies post-exposure prophylaxis and needs to be further evaluated in persons exposed to rabies.
doi:10.4161/hv.20471
PMCID: PMC3551879  PMID: 22699446
rabies; purified chick embryo cell rabies vaccine; purified vero cell rabies vaccine; randomized controlled trial; intradermal rabies vaccination
10.  Cellular immune response following pre-exposure and postexposure rabies vaccination by intradermal and intramuscular routes 
Purpose
Immunization against rabies in humans induces protective neutralizing antibodies; however, the induction of type 1 or type 2 cytokine mediated cellular immune responses following rabies vaccination is not understood. Hence, the present study investigated cellular cytokine responses in vaccinated individuals.
Materials and Methods
The study groups included healthy rabies antigen naive controls (n=10), individuals who received intradermal primary (n=10) or booster pre-exposure vaccination (n=20) and subjects who received postexposure rabies vaccination either by intradermal (n=18) or intramuscular (n=20) routes. The antigen specific cellular responses were analyzed by stimulating peripheral blood mononuclear cells with a rabies vaccine antigen in the interferon-γ (IFN-γ) and interleukin-4 (IL-4) enzyme-linked immunospot (ELISpot) assay. These responses were compared to the rabies virus neutralizing antibody (RVNA) titers that were measured by rapid fluorescent focus inhibition test.
Results
We observed that cellular and humoral immune responses to primary intradermal rabies vaccination could be greatly enhanced by a booster vaccine; and both type 1 and type 2 cytokine responses were significantly elevated. The magnitude of type 1 and type 2 cytokine responses did not differ significantly among the intramuscular and intradermal routes of postexposure vaccination. The number of cells producing IFN-γ and IL-4 correlated significantly with the levels of RVNA.
Conclusion
Both type 1 and type 2 cellular cytokine responses are strongly induced after rabies vaccination and directly correlate with levels of RVNA titers. The neutralizing antibody as well as the type 1 and type 2 cytokine responses may be important for vaccine induced protective responses against rabies.
doi:10.7774/cevr.2015.4.1.68
PMCID: PMC4313111  PMID: 25649188
Rabies; Rabies prophylaxis; Rabies vaccines; IFN gamma; IL4; Immune response
11.  Eliminating Rabies in Estonia 
The compulsory vaccination of pets, the recommended vaccination of farm animals in grazing areas and the extermination of stray animals did not succeed in eliminating rabies in Estonia because the virus was maintained in two main wildlife reservoirs, foxes and raccoon dogs. These two species became a priority target therefore in order to control rabies. Supported by the European Community, successive oral vaccination (OV) campaigns were conducted twice a year using Rabigen® SAG2 baits, beginning in autumn 2005 in North Estonia. They were then extended to the whole territory from spring 2006. Following the vaccination campaigns, the incidence of rabies cases dramatically decreased, with 266 cases in 2005, 114 in 2006, four in 2007 and three in 2008. Since March 2008, no rabies cases have been detected in Estonia other than three cases reported in summer 2009 and one case in January 2011, all in areas close to the South-Eastern border with Russia. The bait uptake was satisfactory, with tetracycline positivity rates ranging from 85% to 93% in foxes and from 82% to 88% in raccoon dogs. Immunisation rates evaluated by ELISA ranged from 34% to 55% in foxes and from 38% to 55% in raccoon dogs. The rabies situation in Estonia was compared to that of the other two Baltic States, Latvia and Lithuania. Despite regular OV campaigns conducted throughout their territory since 2006, and an improvement in the epidemiological situation, rabies has still not been eradicated in these countries. An analysis of the number of baits distributed and the funding allocated by the European Commission showed that the strategy for rabies control is more cost-effective in Estonia than in Latvia and Lithuania.
Author Summary
This paper reports the strategy of oral rabies vaccination of wildlife in Estonia, the measures undertaken to check the method's efficacy and the results obtained. Initiated in autumn 2005, oral vaccination programmes resulted in a dramatic decrease in rabies incidence. All the recommended tests were regularly applied, including the systematic testing of vaccine baits prior to release in the field, serological testing and bait uptake assessment in adult and young animals as well as the typing of all rabies virus isolates. The disease was completely controlled by March 2008, with only three cases reported in summer 2009 and one case in January 2011 in areas very close to the South-Eastern border. The costs associated with rabies control have been calculated and compared on a similar basis for the three Baltic countries. The example of rabies control in Estonia shows that rabies can be quickly and successfully eliminated through successive oral vaccination campaigns by strictly following the recommendations of international organisations. These recommendations concern general strategy, vaccination method and choice of vaccine. To our knowledge, this is the first study showing extensive data from a rabies control programme. The underlying strategy, leading to rabies elimination, is advantageous in terms of cost/effectiveness.
doi:10.1371/journal.pntd.0001535
PMCID: PMC3289618  PMID: 22393461
12.  Susceptibility and lack of evidence for a viremic state of rabies in the night owl monkey, Aotus nancymaae 
Virology Journal  2012;9:95.
Background
Rabies causes an acute fatal encephalomyelitis in most mammals following infection with rhabdovirus of the genus Lyssavirus. Little is known about rabies virus infection in species of New World non-human Primates (NHP). To investigate the suitability of the owl monkey Aotus nancymaae asissue sections examined were unremarkable for inflammation or other histologic signs of rabies a viable animal model for rabies virus candidate vaccine testing, we used clinical presentation, serology, viral isolation, and PCR to evaluate the incubation period, immunity, and pathogenesis of infected animals. We tested the hypothesis that no viremic state exists for rabies virus.
Methods
Eight monkeys divided into two equal groups were inoculated intramuscularly either in the neck or footpad with 105 pfu of rabies virus (Pasteur/V-13R) and observed for >130 days. Oral and blood samples were collected and analyzed.
Results
Two monkeys inoculated in the neck displayed classic paralytic rabies. The mean incubation period was 11.5 days. The average maximum IgG response (antibody titer >0.200 O.D.) was achieved at day 10.0 and 62.3 in the clinical rabies and non-clinical rabies cases, respectively (p = 0.0429). No difference in IgM or IgG time to seroconversion or average maximum IgM level was observed between neck versus footpad inoculation groups. No viremia or viral shedding was detected by PCR or viral isolation during the observation period, including within the two symptomatic animals three days after disease onset. Tissue sections examined were unremarkable for inflammation or other histologic signs of rabies within the asymptomatic animal. Similarly none of the brain sections exhibited immunoreactivity for rabies virus antibody.
Discussion
This study demonstrates there is no difference in time to immune response between inoculation sites and distance to the brain; however, immune response tends to be more rapid in cases of clinically apparent disease and prolonged in cases infected at sites further from the brain.
Conclusions
Our findings support the hypothesis that a viremic state for rabies does not exist in the New World Monkey, Aotus nancymaae, and it appears that this species may be refractory to infection. The species does provide a suitable model to assess post infection immune responses. Additional studies that address the limitations of sample size, length of observation, and lack of measurable infection should be conducted.
doi:10.1186/1743-422X-9-95
PMCID: PMC3522049  PMID: 22612895
Rabies; Rhabdovirus; Lyssavirus; Incubation period; Viremia; Monkey; Aotus nancymaae; Non-human Primate; Ante mortem; Vaccine
13.  Implementation of an Intersectoral Program to Eliminate Human and Canine Rabies: The Bohol Rabies Prevention and Elimination Project 
Background
The province of Bohol, located in the Visayas islands region in the Philippines has a human population of 1.13 million and was the 4th highest region for human rabies deaths in the country, averaging 10 per year, prior to the initiation of the Bohol Rabies Prevention and Elimination Project (BRPEP).
Aims
The BRPEP was initiated in 2007 with the goal of building a sustainable program that would prevent human rabies by eliminating rabies at its source, in dogs, by 2010. This goal was in line with the Philippine National Rabies Program whose objective is to eliminate rabies by 2020.
Methods
The intersectoral BRPEP was launched in 2007 and integrated the expertise and resources from the sectors of agriculture, public health and safety, education, environment, legal affairs, interior and local government. The program included: increasing local community involvement; implementing dog population control; conducting mass dog vaccination; improving dog bite management; instituting veterinary quarantine; and improving diagnostic capability, surveillance and monitoring. Funding was secured from the national government, provincial, municipal and village units, dog owners, NGOs, the regional office of the WHO, the UBS Optimus Foundation, and the Global Alliance for Rabies Control. The BRPEP was managed by the Bohol Rabies Prevention and Eradication Council (BRPEC) under the jurisdiction of the Governor of Bohol. Parallel organizations were created at the municipal level and village level. Community volunteers facilitated the institution of the program. Dog population surveys were conducted to plan for sufficient resources to vaccinate the required 70% of the dogs living in the province. Two island-wide mass vaccination campaigns were conducted followed by “catch up” vaccination campaigns. Registration of dogs was implemented including a small fee that was rolled back into the program to maintain sustainability. Children were educated by introducing rabies prevention modules into all elementary schools in Bohol. Existing public health legislation at the national, provincial, and municipal level strengthened the enforcement of activities. A Knowledge, Attitude and Practices (KAP) survey was conducted in 2009 to evaluate the educational knowledge of the population. Increased surveillance was instituted to ensure that dogs traveling into and out of the province were vaccinated against rabies. Human and animal cases of rabies were reported to provincial and national authorities.
Key Results
Within the first 18 months of the BRPEP, human rabies deaths had decreased annually from 0.77 to 0.37 to zero per 100,000 population from 2007–2009. Between October 2008 and November 2010 no human and animal cases were detected. Increased surveillance on the island detected one suspected human rabies case in November 2010 and one confirmed case of canine rabies in April 2011. Two mass vaccination campaigns conducted in 2007 and 2008 successfully registered and vaccinated 44% and 70% of the dogs on the island. The additional surveillance activities enabled a mobilization of mop up vaccination activities in the region where the human and canine case was located. Due to the increased effective and continuous surveillance activities, rabies was stopped before it could spread to other areas on the island. The program costs totaled USD 450,000. Registration fees collected to maintain the program amounted to USD 105,740 and were re-allocated back into the community to sustain the program.
Author Summary
The Province of Bohol, Philippines has eliminated dog and human rabies in less than three years by empowering the community and implementing an intersectoral strategy. In 2006, Bohol ranked 4th highest in the Philippines for human rabies, averaging 10 deaths per year. Launched in 2007, the program utilized a social awareness campaign, dog population control, mass dog vaccination campaigns, improved dog bite management and veterinary quarantine, a new diagnostic laboratory, expanded surveillance, and the inclusion of education modules into the school curriculum. Improving community compliance to existing national and provincial rabies laws and engaging volunteers to help conduct the project was a key to success. The program, led by the Governor of Bohol, was administered through a group of departments working together at a provincial and local level, and supervised through the Office of the Provincial Veterinarian. Financial support came through the Governor and several NGOs including the Global Alliance for Rabies Control. The program is self-sustaining, through a small dog registration fee fed back into the program, through the continuing education of children in their classrooms, and through the dedicated efforts of over 15,000 staff and volunteers throughout the island.
doi:10.1371/journal.pntd.0001891
PMCID: PMC3516573  PMID: 23236525
14.  Safety and Reactogenicity of an MSP-1 Malaria Vaccine Candidate: A Randomized Phase Ib Dose-Escalation Trial in Kenyan Children 
PLoS Clinical Trials  2006;1(7):e32.
Objective:
Our aim was to evaluate the safety, reactogenicity, and immunogenicity of an investigational malaria vaccine.
Design:
This was an age-stratified phase Ib, double-blind, randomized, controlled, dose-escalation trial. Children were recruited into one of three cohorts (dosage groups) and randomized in 2:1 fashion to receive either the test product or a comparator.
Setting:
The study was conducted in a rural population in Kombewa Division, western Kenya.
Participants:
Subjects were 135 children, aged 12–47 mo.
Interventions:
Subjects received 10, 25, or 50 μg of falciparum malaria protein 1 (FMP1) formulated in 100, 250, and 500 μL, respectively, of AS02A, or they received a comparator (Imovax® rabies vaccine).
Outcome Measures:
We performed safety and reactogenicity parameters and assessment of adverse events during solicited (7 d) and unsolicited (30 d) periods after each vaccination. Serious adverse events were monitored for 6 mo after the last vaccination.
Results:
Both vaccines were safe and well tolerated. FMP1/AS02A recipients experienced significantly more pain and injection-site swelling with a dose-effect relationship. Systemic reactogenicity was low at all dose levels. Hemoglobin levels remained stable and similar across arms. Baseline geometric mean titers were comparable in all groups. Anti-FMP1 antibody titers increased in a dose-dependent manner in subjects receiving FMP1/AS02A; no increase in anti-FMP1 titers occurred in subjects who received the comparator. By study end, subjects who received either 25 or 50 μg of FMP1 had similar antibody levels, which remained significantly higher than that of those who received the comparator or 10 μg of FMP1. A longitudinal mixed effects model showed a statistically significant effect of dosage level on immune response (F3,1047 = 10.78, or F3, 995 = 11.22, p < 0.001); however, the comparison of 25 μg and 50 μg recipients indicated no significant difference (F1,1047 = 0.05; p = 0.82).
Conclusions:
The FMP1/AS02A vaccine was safe and immunogenic in malaria-exposed 12- to 47-mo-old children and the magnitude of immune response of the 25 and 50 μg doses was superior to that of the 10 μg dose.
Editorial Commentary
Background: Malaria is thought to kill between 1 and 2 million people each year in sub-Saharan Africa; most of these are young children under the age of five, who are particularly prone to developing clinical malaria because their immunity is not yet developed. Many groups of researchers around the world are developing candidate vaccines of different types that it is hoped would protect against malaria. One of these types is a “blood-stage” vaccine, which would prevent parasite multiplication in red blood cells. A candidate blood-stage vaccine is FMP1/AS02A, which is designed to raise an immune response against a particular protein (merozoite surface protein-1) on the surface of the blood-stage infectious form of the malaria parasite. In early-stage clinical trials performed in people not exposed to malaria (healthy volunteers in the United States) and in African adults who were exposed to malaria, this candidate vaccine has already been shown to be safe and to bring about an immune response. As part of the next stage in developing this vaccine, a group of researchers next wanted to see whether the vaccine was also safe and brought about an immune response in the population most in need of a vaccine: young children living in an African region with very intense malaria transmission. Therefore, as reported here, this group performed a small trial in western Kenya, recruiting 135 children under 5 y of age to receive either the FMP1/AS02A vaccine (at three different doses) or rabies vaccine for comparison (thus ensuring that children in the control arm got some benefit from being in the trial). The outcomes that the researchers were interested in were primarily adverse events, which they categorized using a standard questionnaire at up to 7 d after vaccination; unsolicited events reported up to 30 d after vaccination; and, finally, any serious events occurring up to 8 mo later. The researchers also examined antibody responses to the FMP1/AS02A vaccine.
What this trial shows: Participants who received the FMP1/AS02A vaccine (as compared to the rabies vaccine) experienced more immediate symptoms, such as pain and swelling at the injection site. Most participants reported unsolicited events during follow-up, but the proportion of participants with adverse events did not seem to be different between the FMP1/AS02A vaccine groups and the rabies vaccine group. Unsolicited outcomes that were reported included, for example, clinical malaria, upper respiratory tract infections, and a few events that were thought to be related to the vaccines, such as fever and eczema. A few serious adverse events occurred up to 8 mo after vaccination, but the numbers did not seem to be different between the FMP1/AS02A and rabies vaccine groups, and the events were not judged to be related to vaccination. Finally, participants who received the FMP1/AS02A vaccine raised an antibody response to the vaccine, which was highest in those who received the highest vaccine dose. The researchers concluded that this vaccine was safe and brought about an immune response in the group of malaria-exposed children studied.
Strengths and limitations: The trial was conducted in a population that is likely to benefit from the vaccine, if it is shown to be effective in further studies. Therefore, the data obtained from this study will be informative in helping to design future trials on FMP1/AS02A. The randomization procedures used in this study were appropriate, and in particular participants of different ages were equally distributed to the different intervention groups, helping to minimize bias. Procedures were also set up to prevent participants and staff giving the vaccines and collecting data from knowing which interventions participants had received. However, the number of participants recruited into the trial was small, and it therefore was not powered to detect anything other than large differences in rates of adverse events between the study groups.
Contribution to the evidence: This study extends evidence from prior trials on the safety and immunogenicity of the FMP1/AS02A vaccine to a population that is representative of those most in need of an effective vaccine—young African children. The results suggest that the vaccine candidate should undergo further evaluation in trials examining vaccine efficacy in a similar population.
doi:10.1371/journal.pctr.0010032
PMCID: PMC1851726  PMID: 17124529
15.  Sickness and recovery of dogs challenged with a street rabies virus after vaccination with a vaccinia virus recombinant expressing rabies virus N protein. 
Journal of Virology  1992;66(5):2601-2604.
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.
PMCID: PMC241012  PMID: 1560518
16.  Rabies Post-Exposure Prophylaxis of Overseas Travelers in the International Travel Clinic of the National Medical Center from 2006 to 2012, Korea 
Infection & Chemotherapy  2014;46(1):13-20.
Background
Rabies is an acute fatal viral disease generally transmitted from infected animals to humans through bites. It is distributed worldwide. The number of Korean people traveling to rabies-endemic countries and being bitten by infected animals has been increasing recently. Therefore, we investigated international travelers who received rabies post-exposure prophylaxis (PEP) at the National Medical Center (NMC) and compared the data with those of other clinics.
Materials and Methods
This study was a retrospective review of 106 patients who visited the International Travel Clinic of the NMC and received rabies PEP between July 2006 and December 2012. During that period, we used the Essen intramuscular regimen protocol. Complete rabies PEP was defined as 5 doses of rabies vaccination with or without rabies immunoglobulin (RIG) administration according to the World Health Organization guidelines.
Results
A total 106 cases documented within the period of 6 years were selected, including 10 children younger than 15 years and 96 older than 15 years. The mean age of the patients who received PEP was 33.4 years. Of the patients, 53 were male and another 53 were female. Most of the exposures occurred in Southeast Asia, predominantly from dog bites (71, 66.9%). The lower extremities were the most frequent site of exposure (37, 34.9%). All the patients began receiving rabies vaccination for prophylaxis after exposure, and 51 received rabies vaccination with RIG. Meanwhile, 74 cases (69.8%) initiated rabies vaccination overseas, but only 10 of them received RIG while overseas; the remaining 32 (30.2%) initiated rabies vaccination after returning to Korea. Within 7 days, all the children and 74 adults received their first rabies vaccination. Six adults initiated first rabies vaccination after 1 week. Eleven of the 106 patients stopped PEP before 5 doses, among whom 4 (1 child and 3 adults) discontinued vaccination after confirming that the biting animal remained healthy throughout 10 days of observation. None of the patients had been previously vaccinated against rabies.
Conclusions
Most of the overseas travelers who visited our clinic after being bitten by suspected rabid animals received appropriate rabies PEP. However, the interval between exposure and first rabies vaccination was often delayed. Tourists who plan to travel in rabies enzootic regions need to be aware that prompt initiation of PEP is important to reduce the risk for developing human rabies.
doi:10.3947/ic.2014.46.1.13
PMCID: PMC3970303  PMID: 24693465
Rabies; Post-exposure prophylaxis; Vaccination
17.  Rabies Risk: Difficulties Encountered during Management of Grouped Cases of Bat Bites in 2 Isolated Villages in French Guiana 
In French Guiana, from 1984 to 2011, 14 animal rabies cases and 1 human rabies case (2008) were diagnosed. In January 2011, vampire-bat attacks occurred in 2 isolated villages. In mid-January, a medical team from the Cayenne Centre for Anti-Rabies Treatment visited the sites to manage individuals potentially exposed to rabies and, in April, an anti-rabies vaccination campaign for dogs was conducted. Twenty individuals were bitten by bats in 1 month, most frequently on the feet. The median time to start management was 15 days. The complete Zagreb vaccination protocol (2 doses on day 0 and 1 dose on days 7 and 21) was administered to 16 patients, 12 also received specific immunoglobulins. The antibody titration was obtained for 12 patients (different from those who received immunoglobulins). The antibody titers were ≥0.5 EU/mL for all of them. The serology has not been implemented for the 12 patients who received immunoglobulins. Accidental destruction of a vampire-bat colony could be responsible for the attacks. The isolation and absence of sensitization of the populations were the main explanations for the management difficulties encountered. Sensitization programs should be conducted regularly.
Author Summary
Rabies is a disease almost invariably fatal in humans once the first clinical signs appear. In French Guiana bats represent the virus reservoir, especially vampire bats. From 1984 to 2011, 14 animal rabies cases and 1 human rabies case (2008) were diagnosed. In case of bat bite, anti-rabies immunoglobulins (RIG) and rabies vaccine must be rapidly administrated. The specific rabies management is exclusively performed by Centre for Anti-Rabies Treatment (CART), located at the Institut Pasteur in Cayenne, the prefecture of French Guiana, and 6 Anti-Rabies Treatment Outposts distributed along the coastal edge and along the two main rivers. Only a CART physician can administer RIG. In January 2011, vampire-bat attacks occurred in 2 isolated villages. In mid-January, a medical team from the CART visited the sites to manage individuals potentially exposed to rabies and, in April, an anti-rabies vaccination campaign for dogs was conducted. The most relevant contribution of this study is to underline difficulties to provide rabies post-exposure prophylaxis to remote populations exposed to bat rabies in the Amazonian region and to show the lack of awareness of these rural populations concerning rabies and the risk associated to vampire bats.
doi:10.1371/journal.pntd.0002258
PMCID: PMC3694830  PMID: 23826400
18.  Purified chick embryo cell rabies vaccine: economical multisite intradermal regimen for post-exposure prophylaxis. 
Epidemiology and Infection  1987;99(3):755-765.
The standard six-dose intramuscular (i.m.) rabies post-exposure vaccine regimen using a new purified chick embryo cell (PCEC) vaccine was compared with two economical multisite intradermal (i.d.) PCEC regimens, a multisite i.m. PCEC schedule and a subcutaneous regimen using a suckling mouse brain (SMB) rabies vaccine manufactured in Thailand. The neutralizing antibody results for the four-site and eight-site i.d. and the standard i.m. PCEC regimens were similar over 3 months. A three-site i.m. PCEC regimen had no advantage. The SMB vaccine gave the lowest antibody levels. Human rabies immune globulin therapy significantly increased the GMT of all groups on day 7, unlike equine antirabies serum (EARS). Both antisera suppressed antibody responses to PCEC on days 14 and 28. Three generalized reactions probably related to EARS were the only serious side effects. An eight-site i.d. PCEC vaccine regimen proved as immunogenic as the routine i.m. schedule and, if implemented as post-exposure prophylaxis, would be the cheapest widely available tissue culture vaccine regimen. The protective efficiency should now be tested in patients bitten by rabid animals.
Images
PMCID: PMC2249234  PMID: 3428378
19.  Antibody Response to a Human Diploid Cell Rabies Vaccine 
Applied Microbiology  1974;27(3):553-561.
An experimentally killed rabies virus vaccine prepared in a human diploid cell strain (WI-38)—Wyeth rabies vaccine (WRV)—was used by various injection schedules in two separate studies to define more closely in human volunteer subjects an effective vaccination schedule for pre- or postexposure immunization, particularly for donors of rabies-hyperimmune plasma. To permit valid comparisons between our results and those of other workers, antibody levels achieved were expressed in terms of international units (IU) per milliliter of serum. Antibody response of previously nonvaccinated persons were only modest after a single dose of WRV, never reaching a level higher than 0.80 IU/ml over a 56-day testing period. Moreover, antibody was not detected at 0.16 IU/ml before the 14th day, either after a single dose or after two doses given 3 days apart. The best response followed four doses of WRV given within 4 weeks. This schedule resulted in the highest rate of seroconversion to the ≥6 IU/ml antibody level required of potential rabies-immune plasma donors. Giving the first vaccine dose in aluminum hydroxide diluent did not enhance the antibody response. There was a definite suggestion in the various injection schedules that higher and more sustained antibody levels were reached when the interval between the first and second vaccine doses was longest. The greater immunogenicity of WRV as compared with duck embryo vaccine was best demonstrated by the fact that a single booster dose of duck embryo vaccine to previously vaccinated individuals resulted in only a sevenfold antibody rise during the following 56 days, whereas a booster dose of WRV elicited a 69-fold rise. Al(OH)3 in the first dose of WRV had no effect, but the enhancing effect of a longer interval between vaccine doses was noted once again; 20 of 20 subjects who received three doses of WRV with 4 weeks between doses developed good levels of rabies antibody, and 19 exceeded 6 IU/ml.
PMCID: PMC380083  PMID: 4856855
20.  Comparison of Safety and Immunogenicity of PVRV and PCECV Immunized in Patients with WHO Category II Animal Exposure: A Study Based on Different Age Groups 
Background
The aim of this study was to compare the safety and immunogenicity between purified vero cell rabies vaccine (PVRV) and purified chick embryo cell vaccine (PCECV) in patients with WHO category II animal exposure, especially in different age groups.
Methodology/Principal Findings
In one-year clinical observation after vaccination with PVRV or PCECV under Zagreb (2-1-1) or Essen (1-1-1-1-1) regimens, information collection for the demographic and adverse events (AEs) and rabies virus laboratory examination of neutralizing antibody (RVNA) titers were performed for all patients with WHO category II animal exposure in Wuhan city. The results showed no significant differences of safety and immunogenicity between PVRV and PCECV both in Zagreb and Essen regimens. However, when compared with other age groups, most systemic AEs (36/61) occurred in <5-year-old patients, and <5-year-old patients have significant lower RVNA titer and seroconversion rate (RVNA ≥0.5 IU/ml) at day 7 both in Zagreb and Essen regimens or PVRV and PCECV groups.
Conclusions
Our data showed that vaccination with PVRV is as safe and immunogenic as PCECV in patients of all age groups, but might be more popular for clinical use. When performing a vaccination with rabies vaccine in young children, the most optimal vaccine regimen should be selected.
Author Summary
Nowadays, many approved vaccines with different components (such as purified vero cell rabies vaccine [PVRV], purified chick embryo cell vaccine [PCECV], and Human diploid cell vaccine [HDCV]) and many regimens with different vaccination schedules (Zagreb, Essen) are being used in the world. Thus, we compared the safety and immunogenicity between purified vero cell rabies vaccine (PVRV) and purified chick embryo cell vaccine (PCECV) in patients with WHO category II animal exposure, especially in different age groups. Our data showed no significant differences of safety and immunogenicity between PVRV and PCECV with Zagreb or Essen regimen in four age groups. However, compared with the other three age groups, young children aged less than 5 years have more systemic adverse events (AEs), and lower rabies virus neutralizing antibody (RVNA) titer and seroconversion rate (RVNA ≥0.5 IU/ml) at day 7 post-immunization. These findings highlight that it is important for young children, a population with more than 50% of human rabies deaths, to find the most optimal vaccine and vaccination schedule in the future.
doi:10.1371/journal.pntd.0003412
PMCID: PMC4270726  PMID: 25522244
21.  Evaluation of a new five-injection, two-site,intradermal schedule for purified chick embryo cell rabies vaccine: A randomized, open-label, active-controlled trial in healthy adult volunteers in India 
Background:
Human rabies is an ongoing significant public health problem inmany developing countries, with India reporting the highest incidence of rabies-related deaths (∼20,000 per year). Many people living in India cannot afford the standard IM postexposure prophylaxis (PEP) with cell-culture vaccines, which are administered using a 5-dose regimen developed in Essen, Germany. A potentially less expensive intradermal (ID) regimen, based on the Essen regimen, has been developed at the Kempegowda Institute of Medical Sciences (KIMS), Bangalore, India.
Objective:
The objective of this study was to compare the immunogenicity and local and systemic tolerability of the KIMS-1D regimen with those of the standard Essen IM regimen in healthy adult volunteers in India.
Methods:
This randomized, open-label, active-controlled trial was conductedat the Antirabies Clinic, Medical College, KIMS. Healthy adult volunteers were randomly assigned to receive purified chick embryo cell vaccine (PCECV) using the KIMS-1D regimen (0.1 mL injected ID at 2 body sites on days 0, 3, 7, 14, and 28 [“2-2-2-2-2”]) or the Essen IM regimen (1 mL injected IM at 1 body site on the same days Subjects were followed up for 365 days by the treating physician and encouraged to voluntarily report any adverse events (AEs). Serum rabies virus-neutralizing antibody (RVNA) concentrations were measured before the first injection on day 0 (baseline) and on days 14, 28, 90, 180, and 365, using the rapid fluorescent focus inhibition test.
Results:
Ninety-one subjects were enrolled and included in the tolerabilityand immunogenicity analyses. The ID group comprised 45 subjects (26 men, 19 women; mean [SD] age, 20.84 [1.48] years); the IM group, 46 subjects (28 men, 18 women; mean [SD] age, 21.02 [1.16] years). The most common local AEs were pain at the injection site (2/225 [0.9%] in the ID group and 10/230 [4.3%] in the IM group; P < 0.006) and itching at the injection site (5/225 [2.2%] in the ID group and none in the IM group; P = 0.026). All of the AEs were transient and resolved without the need for medication. All subjects had serum RVNA concentrations ≥0.5 IU/mL—considered protective by the World Health Organization—at all follow-up visits. However, the mean RVNA concentrations in the IM group were significantly higher compared with those in the ID group from days 14 to 365 (all, P < 0.001).
Conclusion:
In this study in healthy volunteers, PEP with PCECV administered using the KIMS-ID regimen was well tolerated and immunologically efficacious for 365 days. Adequate RVNA levels were maintained with the KIMS-ID regimen from days 14 to 365, although these levels were significantly lower than those achieved in the group receiving the Essen IM regimen (all, P < 0.001).
doi:10.1016/j.curtheres.2005.08.009
PMCID: PMC3964532  PMID: 24672132
rabies; intradermal rabies vaccination; KIMS-ID regimen; randomizedcontrolled trial
22.  Survey of fox trappers in northern Alaska for rabies antibody. 
Epidemiology and Infection  1994;113(1):137-141.
The purpose of this research was to determine whether trappers in northern Alaska acquired immunity to rabies virus from non-bite exposures while trapping and skinning arctic foxes (Alopex lagopus). In coastal Alaska recurring epizootics presumably provide trappers ample opportunity for contact with rabid animals. Serum neutralization analyses of blood samples collected from 26 individuals were conducted. All but three had negative rabies neutralizing antibody levels (< 0.05 I.U./ml). Two of these had previously received rabies vaccine but one individual who had trapped for about 47 years with an estimated harvest of over 3000 foxes and who had never received pre- or post-exposure rabies vaccination had a rabies serum neutralizing antibody concentration of 2.30 I.U./ml. This represents the first report of an unvaccinated person acquiring rabies virus antibody with a titre above the 0.5 I.U./ml level considered acceptable by the World Health Organization.
PMCID: PMC2271209  PMID: 8062870
23.  Cell Culture Extraction and Purification of Rabies Virus Nucleoprotein 
Background:
Rabies is a major zoonotic viral disease and is detected using the World Health Organization standard diagnostic techniques. Rabies detection is preferably done using the fluorescent antibody technique (FAT) that provides reliable diagnosis with almost 100% accuracy for all variant strains, if a proper conjugate is used. Rabies virus nucleoprotein (NP) is the most important protein used in production of a specific diagnostic conjugate.
Objectives:
The aim of this study was to extract the cell-associated rabies virus NP from infected Baby Hamster Kidney cell clone (BSR) with rabies virus (Pasteur vaccine strain/PV) and purify for a future project to produce an anti-NP conjugate.
Materials and Methods:
Pasteur vaccine strain (PV) as the standard rabies vaccine strain with a focus-forming dose (FFD) of 105 was inoculated in to the BSR cell culture at a concentration of 106 cells per milliliter. Infected cells were harvested 72 hours after infection and the rabies NP was extracted from these cells by low-speed centrifugation and purification by ultracentrifugation in cesium chloride (CsCl) gradient. For analysis, the purified NP was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
Results:
The volume of the lysate was 15 mL and it became 2.5 mL after purification, with a concentration of 3.25 mg/mL. The corresponding band to the cell lysate protein on the SDS-PAGE had a molecular weight of 50 KDa, similar to the molecular weight of NP in rabies virus.
Conclusions:
The rabies virus NP could be extracted and purified in an appropriate amount from infected cell culture. The results of SDS-PAGE analysis showed that the intact rabies virus NP had been purified properly and thus could be used for further steps to produce the specific diagnostic rabies conjugate.
doi:10.5812/jjm.11734
PMCID: PMC4255371  PMID: 25485056
Fluorescent Antibody Technique; Diagnosis; Rabies Virus; Isolation and Purification; Nucleoproteins
24.  Potential and Actual Terrestrial Rabies Exposures in People and Domestic Animals, Upstate South Carolina, 1994–2004: A Surveillance Study 
BMC Public Health  2009;9:65.
Background
Although there has been a reduction of rabies in pets and domestic animals during recent decades in the United States, rabies remains enzootic among bats and several species of terrestrial wildlife. Spillover transmission of wildlife rabies to domestic animals therefore remains a public health threat
Methods
Retrospective analysis of surveillance data of reported animal incidents (bites, scratches, mucous membrane contacts) from South Carolina, 1995 to 2003, was performed to assess risk factors of potential rabies exposures among human and animal victims.
Results
Dogs and cats contributed the majority (66.7% and 26.4%, respectively) of all reported incidents, with stray dogs and cats contributing 9.0% and 15.1 respectively. Current rabies vaccination status of dogs and cats (40.2% and 13.8%, respectively) were below World Health Organization recommended levels. Owned cats were half as likely to be vaccinated for rabies as dogs (OR 0.53, 95% CI 0.48, 0.58). Animal victims were primarily exposed to wildlife (83.0%), of which 27.5% were rabid. Almost 90% of confirmed rabies exposures were due to wildlife. Skunks had the highest prevalence of rabies among species of exposure animals (63.2%). Among rabid domestic animals, stray cats were the most commonly reported (47.4%).
Conclusion
While the majority of reported potential rabies exposures are associated with dog and cat incidents, most rabies exposures derive from rabid wildlife. Stray cats were most frequently rabid among domestic animals. Our results underscore the need for improvement of wildlife rabies control and the reduction of interactions of domestic animals, including cats, with wildlife.
doi:10.1186/1471-2458-9-65
PMCID: PMC2651164  PMID: 19236696
25.  The Activity of Rabies Vaccines against Genetic Clusters of Rabies Virus Circulating at the Territory of Ukraine 
Objective
To identify the presence of genetic clusters of rabies virus at the territory of Ukraine and to determine the degree of activity of rabies vaccines against these genetic clusters.
Introduction
To develop and implement an effective program of rabies eradication in Ukraine in 2008 was founded the unique collection of samples of pathological materials confirmed as positive in rabies at the regional veterinary laboratories of Ukraine. The collection is constantly updated and to present moment it includes 1389 samples from all regions of Ukraine, selected from 17 animal species and humans.
Methods
Identification of the rabies virus in samples of pathological material for their further selection was carried out using the test developed by us which based on RT-PCR with primers complementary to the conservative fragments of the 5’-end of nucleoprotein gene of rabies virus.
For the study of the street rabies virus isolates from the collection we use RT-PCR with the primers pair (509, 304) flanking the variable 3’-end part of nucleoprotein gene of the reference strain of rabies virus CVS (fragment in 377 bp).
Studies of rabies vaccines activity were carried out with modified method of U.S. National Institutes of Health using rabies virus street isolates of both genetic clusters instead of the Challenge Virus Standard (CVS). All isolates of street rabies virus were inoculated in a dose of 5–50 LD50. The criteria for evaluation of protective activity of rabies vaccine was effective dose (− lg ED50).
Results
In molecular genetic studies with variant-specific primers we established the presence in Ukraine of two clusters of rabies virus. Clusters I circulates on the right bank of the Dnipro river (the largest water barrier that divides the country into eastern and western side), and cluster II – on the left bank of the Dnieper.
The relationship of these variants with the epizootic situation was researched. For this purpose epizootological zoning of Ukraine according to the intensity of the epizootic situation in 2005–2009 was carried out. As a result of this analysis all the regions of Ukraine belong to three categories: high, medium and low epizootic situation intensity of rabies.
The projection of differentiated genetic clusters on the epizootic situation showed that cluster II circulating at Left Bank of the Dnieper in areas with high and medium intensity of the epizootic situation, and the cluster I – at the Right Bank of the Dnieper, mainly in the areas with low intensity of the epizootic situation.
That’s why our interest was in the degree of protection of rabies vaccines against street rabies virus isolates belonging to these two genetic clusters.
The commercial vaccines made with rabies virus vaccine strains SAD (Street-Alabama-Dufferin) and Wistar PM/WI were chosen to evaluate this parameter.
After the mathematical calculations of effective dose and the analysis of the data the less effective protection of rabies vaccines (at 29–30 %) against street rabies virus isolates belonging to cluster II in comparison with isolates belonging to cluster I irrespective to the strain vaccine is made was shown.
Conclusions
The data will be used for the effective planning of specific prophylaxis of rabies in Ukraine based on differentiated approach to distribution of rabies vaccines in according to region and their activity.
PMCID: PMC3692803
rabies vaccine; vaccine activity; street rabies virus isolates; genetic variants of rabies virus

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