Field trials of a very potent rabies DNA vaccine which induced long lasting virus neutralizing antibodies and protection in dogs in experimental conditions

Kinetics of antibody responses and protection against rabies were investigated after injection of a single dose of rabies DNA vaccine and compared to those induced by one or two injections of cell culture-derived vaccine in dogs issued from the common local breed and reared in experimental conditions. Rabies DNA vaccine administered intradermally by a jet injector in the inner face of the ear was by far more efficient in inducing long lasting high titers of virus neutralizing antibodies compared to cell culture vaccine Rabisin administered either subcutaneously or intramuscularly. Four years after vaccine administration of either DNA or cell culture-derived rabies vaccines, full protection against a rabies peripheral challenge was achieved. Vaccine trials targeting dogs living in field conditions in Tunisia further established that rabies DNA-based vaccination induced a stronger induction of virus neutralizing antibodies compared to Rabisin. This report shows for the first time that DNA vaccination could be more efficient under experimental or field conditions in large size mammals than the best commercially available cell culture-derived vaccine. This improvement will hopefully allow a better rabies control in developing countries by using a more efficient vaccination with fewer doses and targeting all categories of dogs.     

Three-year duration of immunity in cats vaccinated with a canarypox-vectored recombinant rabies virus vaccine

Despite the availability of efficacious vaccines for animals and humans, rabies is still a major zoonosis. Prevention of rabies in dogs and cats is key for reducing the risk of transmission of this deadly disease to humans. Most veterinary vaccines are adjuvanted inactivated vaccines and have been shown to provide one to four-year duration of immunity. In response to debates about the safety of adjuvanted vaccines in cats, a non-adjuvanted feline rabies vaccine with one-year duration of immunity claim was specifically developed using the canarypoxvirus vector technology. The objective of this study was to validate a vaccination program based on primary vaccination, revaccination one year later and boosters every three years. Seronegative cats were vaccinated at 12 weeks of age and received a booster vaccination one year later. This vaccination regimen induced a strong and sustained antibody response, and all vaccinated animals were protected against virulent rabies challenge carried out 3 years after vaccination.     

Experimental studies on the duration of immunity in dogs vaccinated against rabies

The results of laboratory investigations on the duration of immunity in dogs vaccinated against rabies are summarized; the experiments demonstrated that dogs vaccinated with phenol-treated killed-virus vaccine show a high degree of immunity at one year after inoculation and that the immunity produced with the Flury-strain live-virus vaccine is superior to, and of longer duration than, that obtained with chemically-treated brain-tissue virus vaccine.     

Longevity of neutralizing antibody levels in macaques vaccinated with Quil A-adjuvanted measles vaccine candidates

Quil A-based candidate measles vaccines have been shown to be immunogenic and protective in cotton rats and macaques. Here we studied the longevity of protective VN antibody levels induced in macaques with one dose of measles virus (MV) iscom. Inactivated MV adjuvanted with iscom-matrix or with purified Quillaja saponin QA-22 were also tested. All animals developed high levels of VN antibody and MV-specific IFNgamma-producing cells. Especially the high VN antibody levels induced by the latter two preparations showed virtually no decrease during the 2-year follow-up. These highly promising candidate MV vaccines should now be tested in infant macaques in the presence or absence of passively transferred and/or maternally derived VN antibodies. In addition, the immunopathological safety of the constructs should be evaluated in the atypical measles model in rhesus macaques.     

狂犬病毒中和抗体检测快速荧光灶抑制试验的建立

目的 建立检测狂犬病毒中和抗体的快速荧光灶抑制试验(RFFIT),检测狂犬病毒中和抗体滴度.方法 以狂犬病标准毒株CVS-11为标准攻击毒,制备三代病毒,建立CVS-11病毒库;以国际标准抗狂犬病毒血清为对照血清,参考法国巴斯德研究所的RFFIT操作标准,建立适应于中国狂犬病毒中和抗体检测的RFFIT方法 ,并验证该方法 的特异性、稳定性和重复性.结果 成功建立了检测狂犬病毒中和抗体的RFFIT试验,验证该试验方法 的特异性为100%;重复性试验表明,在本实验室和不同实验室的检测结果 P值均＞0.5,具有很好的重复性.结论 RFFIT方法 的建立进一步完善了中国狂犬病的实验室检测技术,也将提升中国狂犬病的整体监测能力.     

Rabies virus glycoprotein serology ELISA for measurement of neutralizing antibodies in sera of vaccinated human subjects

BackgroundVaccination provides protection against infection by inducing VNAs mainly against RABV surface GP. The measurement of VNAs to RABV is commonly used to assess the level of immunity in humans and animals after vaccination. A VNA titer of ≥ 0.5 IU/mL of sera indicates adequate response to vaccination. Here, we report the development and validation of a RABV GP serology ELISA kit for semi-quantitative measurement of VNA titers in sera of vaccinated human subjects.MethodsUsing a recombinant RABV GP expressed in mammalian cells as the capture antigen, the ELISA method was established using HuMAb NM57 reference initially and HRIG reference subsequently. The limit of detection (LOD), linear range, reproducibility, and precision of the method were examined. Specificity and sensitivity were established to assess the diagnostic accuracy.ResultsRABV GP for ELISA plate coating and optimal dilution of human serum sample was 1 µg/mL and 1:20, respectively. Multiple assays were carried out by different technicians at different laboratories for assay standardization. Using the HRIG reference, the LOD was found to be 0.02–0.06 IU/mL and the linear range was 0.2–10.0 IU/ mL. The inter-assay CVs were in the range of 6.60–10.79%, indicating the reproducibility. None of the 12 known negative human sera, tested positive by ELISA, highlighting the specificity. A total of 415 unknown positive human sera were double-blind tested by the RFFIT and ELISA. The VNA titer cut-off value of ELISA was set at 1.5 IU/mL to ensure no false-positive. The diagnostic specificity and sensitivity were 100% and 91.1%, respectively.ConclusionsThe validation data characterize this ELISA as a suitable method for semi-quantitative measurement of VNA titers in human serum samples to assess vaccination status. The ELISA kit can offer simplicity, speed, low cost and high throughput, making it a practical tool for monitoring the immune response following vaccination.     

Evaluation of an improved rapid neutralizing antibody detection test (RAPINA) for qualitative and semiquantitative detection of rabies neutralizing antibody in humans and dogs

Using the principle of immunochromatography, we previously developed a method called RAPINA (Rapid Neutralizing Antibody detection test) that can measure the level of rabies virus -neutralizing antibody (VNA) in serum samples [Shiota S, Mannen K, Matsumoto T, Yamada K, Yasui T, Takayama K, et al. Development and evaluation of a rapid neutralizing antibody test for rabies. J Virol Methods 2009;161:58-62]. RAPINA is faster, simpler, and easier to perform compared with a virus-neutralizing test or enzyme-linked immunosorbent assay (ELISA). The improved version of RAPINA has greater positive and negative predictive values corresponding to a VNA level of 0.5 IU/mL, as recommended by the World Health Organization and the World Organization for Animal Health. To verify the efficacy of this improved method, serum samples were collected from humans and dogs before and after immunization against rabies and were tested in Japan, Sri Lanka, and Thailand. The results were compared between RAPINA and the true VNA levels measured by the Rapid Fluorescent Focus Inhibition Test (RFFIT). The improved RAPINA accurately predicted seropositivity for 182 of 183 seropositive human samples as assessed by RFFIT (99.5%) and for 138 of 140 RFFIT-negative human samples (98.6%). In dog serum samples, the positive and negative predictive values were 99.7% (345/355) and 95.6% (174/182), respectively. RAPINA was also used to estimate VNA levels in a semiquantitative manner by using serial dilution of serum samples. Our results show that RAPINA is an easy and rapid method for measuring VNA levels before and after immunization with the rabies vaccine and does not need a high skill level or sophisticated equipment. RAPINA can be used to monitor the success of preexposure prophylaxis in at-risk persons, vaccine coverage, and animal control. It can also be used in laboratories with modest facilities and where a large number of samples are screened.     

Predictive factors for the neutralizing antibody response following pre-exposure rabies immunization: validation of a new booster dose strategy

A prospective cohort of 312 subjects who received pre-exposure rabies immunization and who were monitored serologically with a 10-year follow-up was assessed using multivariate analysis. The aim was to propose a new booster dose strategy by identifying predictive factors for the durability of the neutralizing antibody response. Evaluation bore on several factors relating to: (1) demographic characteristics: age, gender; (2) vaccines: type of vaccine (HDCV or PVRV), injection regimen (D0-D28-D365 or D0-D7-D28-D365) and vaccine lots' antigenic potency; and (3) resulting antibody titers. Logistic regression analysis enabled the authors to establish a predictive model for immunized subjects' serological status at ten years' follow-up expressed as a P probability for seroreversion (antibody titer <0.5 IU/ml). Highly significant factors were the immunization regimen, the type of vaccine used and the antibody titer at D379. A P value <0.4 identified subjects as "good" responders who were sure to be have satisfactory antibody titers at 10 years and who required a single booster dose every 10 years. A P value >/=0.4 identified subjects as "poor" responders in whom a specific follow-up and booster dose strategy is proposed. This new immunization strategy could at least be applied to subjects with a frequent risk of exposure, as defined by institutional recommendations. This new immunization strategy should nevertheless undergo an external validation and a cost-effectiveness evaluation.     

Experimental immunization of cats with a recombinant rabies-canine adenovirus vaccine elicits a long-lasting neutralizing antibody response against rabies

During the past decade, human rabies caused by cats has ranked the second highest in China. Several recombinant rabies vaccines have been developed for dogs. However, seldom have these vaccines been assessed or used in cats. In this trial, we report the experimental immunization of a recombinant canine adenovirus-rabies vaccine, CAV-2-E3Delta-RGP, in cats. Thirty cats were inoculated with the recombinant vaccine intramuscularly, orally and intranasally, respectively. Safety and efficacy studies were undertaken using the fluorescent antibody virus neutralization (FAVN) test and evaluated. Results showed that this recombinant vaccine is safe for cats as demonstrated by the three different routes of administration. The vaccine stimulated an efficient humoral response in the vaccinated cats when 10(8.5)PFU/ml of the recombinant vaccine was injected intramuscularly in a single dose. The neutralizing antibody level increased above 0.5IU/ml at 4 weeks after the vaccination. The mean antibody level ranged from 0.96+/-0.26 to 4.47+/-1.57IU/ml among individuals, and the antibody levels were elicited for at least 12 months. After this period, the immunized cats survived the challenge of CVS-24 and an obvious anemnestic and protective immune response was stimulated after the challenge. The immune response occurred later than the inactivated vaccine and the overall antibody level in the vaccinated cats was lower, but it was sufficient to confer protection of cats against infection. This demonstrated that a single, intramuscular dose of CAV-2-E3Delta-RGP stimulated a long-lasting protective immune response in cats and suggested that CAV-2-E3Delta-RGP could be considered as a potential rabies vaccine candidate for cats.     

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

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

No adverse effects of simultaneous vaccination with the immunocontraceptive GonaCon™ and a commercial rabies vaccine on rabies virus neutralizing antibody production in dogs

Parenteral vaccination campaigns are integral to the elimination of canine rabies. To maximize herd immunity in dogs, immunocontraception provided at the time of rabies vaccination should reduce fecundity and dog abundance. GonaCon™ has been used successfully as an immunocontraceptive in a variety of mammals, and by inference, the dog would be an ideal candidate for testing. As an initial step in evaluating a combination-vaccination program, we assessed the effects of GonaCon™ on rabies virus neutralizing antibody production in dogs after administration of a veterinary rabies vaccine. Eighteen feral/free ranging dogs were included in this initial study: six were given GonaCon™ only, six were given rabies vaccination only, and six received GonaCon™ and rabies vaccination. Antibody levels were evaluated over 82 days. The use of the immunocontraceptive GonaCon™ did not affect the ability of dogs to seroconvert in response to the rabies vaccine. Thus, GonaCon™ provides a potential immunocontraceptive for use in combination with rabies vaccine to increase herd immunity and address dog population over abundance to better manage rabies.     

Production of rabies neutralizing antibody in hen's eggs using a part of the G protein expressed in Escherichia coli

In an attempt to produce anti-rabies immunoglobulin affordable for people living in developing countries, we have immunized layer chickens with a part of the G protein of rabies virus expressed in Escherichia coli. Immunoglobulin (IgY) was purified from the yolks of eggs layed by immunized hens. It was revealed in vitro that the antibody specifically bound to virions as well as cells infected with rabies virus. Moreover, the antibody apparently neutralized rabies virus infectivity. Inoculation of the antibody into mice infected with rabies virus reduced the mortality caused by the virus, suggesting that IgY directed to the part of the G protein expressed in E. coli could serve as a possible alternative to currently available anti-rabies human or equine immunoglobulins.     

First administration to humans of a monoclonal antibody cocktail against rabies virus: safety, tolerability, and neutralizing activity

Immediate passive immune prophylaxis as part of rabies post-exposure prophylaxis (PEP) often cannot be provided due to limited availability of human or equine rabies immunoglobulin (HRIG and ERIG, respectively). We report first clinical data from two phase I studies evaluating a monoclonal antibody cocktail CL184 against rabies. The studies included healthy adult subjects in the USA and India and involved two parts. First, subjects received a single intramuscular dose of CL184 or placebo in a double blind, randomized, dose-escalation trial. Second, open-label CL184 (20IU/kg) was co-administered with rabies vaccine. Safety was the primary objective and rabies virus neutralizing activity (RVNA) was investigated as efficacy parameter. Pain at the CL184 injection site was reported by less than 40% of subjects; no fever or local induration, redness or swelling was observed. RVNA was detectable from day 1 to day 21 after a single dose of CL184 20 or 40IU/kg. All subjects had adequate (>0.5IU/mL) RVNA levels from day 14 onwards when combined with rabies vaccine. CL184 appears promising as an alternative to RIG in PEP.     

Canine rabies DNA vaccination: a single-dose intradermal injection into ear pinnae elicits elevated and persistent levels of neutralizing antibody

Rabid dog exposures cause >99% of human rabies deaths world-wide. In developing countries, where dogs are the viral reservoir, the 30-50% vaccination coverage of dog populations is insufficient to break the disease transmission cycle. In addition, many vaccines currently used in developing countries fail to maintain detectable levels of neutralizing antibody. The poor vaccination coverage with inadequate vaccines, in addition to the difficulty in locating dogs for booster vaccinations, suggest that an inexpensive vaccine that elicits long-term immunity after a single-dose vaccination could improve control of canine rabies in developing countries. One solution could be a DNA vaccine. This study was designed to evaluate in dogs the ability of different methods of a single-dose DNA vaccination to elicit enhanced levels of neutralizing antibody. Intradermal (i.d.) vaccination into ear pinnae elicited elevated and long-lasting levels of neutralizing antibody. Minimal or undetectable levels of neutralizing antibody were detected after vaccination into quadriceps muscle, gene gun vaccination into ear pinnae or i.d. vaccination into the neck. Intramuscular (i.m.) or gene gun vaccinations did not "immunologically prime" a majority of dogs vaccinated by these routes. The passive transfer of sera from dogs that had been vaccinated i.d. in ear pinnae protected mice against rabies virus challenge. A single-dose i.d. rabies DNA vaccination into ear pinnae could aid in the control of canine rabies in developing countries.     

Responses to human diploid cell rabies vaccine: neutralizing antibody responses of vaccinees receiving booster doses of human diploid cell rabies vaccine.

Rabies neutralizing antibody levels were determined before and after administration of a booster-dose of Wyeth rabies vaccine (WRV) in persons immunized earlier with either duck embryo vaccine (DEV) or with WRV. Virtually all those receiving an initial 3-dose regimen of WRV (0, 7 and 21--28 days) still had neutralizing antibody one year later, but there was a decline in titer from 10--50 IU per ml at 35 days to about 1--3 IU. Only one-half of those receiving DEV as the primary vaccine had even detectable antibody one year later. All volunteers responded anamnestically to a single WRV booster given 8--12 months after either primary vaccine. Those given WRV initially had much higher antibody levels than those given DEV, but after the WRV booster antibody levels in all vaccinees remained high, even one year later.     

Results of a study on the under-detection of meningococcus in vaccinated subjects in Galicia

BACKGROUND: On the basis of active surveillance and the monitoring of Meningococcal Disease (MD) following the vaccination campaign carried out in Galicia, it was observed that the proportion of isolations of the serogroups responsible for the disease among individuals suspected of Meningococcal Disease (SMD) who had been vaccinated was lower than among unvaccinated individuals. In view of this situation, a study was made in order to determine whether in the origin of those SMDs that were not isolated, we would find N. Meningitidis serogroup C, and to quantify the significance of the sub-detection of same. METHODS: For this purpose, and during the period under study (from the 26th week of 1997 to the 14th week of 1999), blood and cephalorachidian fluid samples were taken from the SMDs without isolation for their study with C protein reagent for type and serogroup. The analysis of the samples was performed by the microbiology laboratory of the Clinical Hospital of Santiago de Compostela. RESULTS: Of the 120 cases notified during the period under study, 65 were analysed by C protein reagent (38 vaccinated and 27 unvaccinated), with a positive reading for N. meningitidis in 65% (42 samples) 74% in vaccinated individuals and 52% in unvaccinated. By estimating, on the basis of the cases studied, the results for the total, and excluding the C protein reagent negative cases, we find that, for serogroup C, in only 27% of the cases occurring in vaccinated individuals was it possible to isolate it, in comparison with 80% in the case of unvaccinated subjects (p < 0.0001). These percentages are, in the case of serogroup B, 59% and 71%, respectively, a difference which is not statistically significant. CONCLUSIONS: The vaccine brought about an true sub-detection of serogroup C meningococci in the vaccinated cases.     

Persistence of antibody response to pneumococcal capsular polysaccharides in vaccinated long term-care residents in Brazil

To evaluate the immunogenicity of 23-valent pneumococcal polysaccharide vaccine in 52 nursing homes residents aged > or = 60 years, IgG antibodies to serotypes 1, 5, 6B, and 8 were measured by ELISA and compared before, and 1 and 12 months following vaccination. A significant immunological response for all serotypes was observed at 1 month after vaccination. The mean increase in antibody concentration was highly variable and ranged from 1.6 to 2.7. After 1 year, the mean concentrations remained significantly higher than prior to vaccination for serotypes 1, 6B, and 8, although there was a decrease in all mean IgG concentrations. Antibody levels were higher in men than in women, before and after immunisation. Post-vaccination values tended to be lower among subjects aged >75 years. Reduction in IgG concentrations by 33% 1 year after vaccination suggests that revaccination of institutionalised elderly people may be needed.     

Falciparum malaria after splenectomy: a prospective controlled study of 33 previously splenectomized Malawian adults

We identified 33 Malawians who had undergone total splenectomy for traumatic injury. We reviewed these and 33 controls by clinical and parasitological examination monthly for 1 year. Splenectomized patients (S) were 2.5 times as likely as controls (C) to complain about febrile symptoms during the month preceding a visit (P < 0.0001). They were nearly twice as likely as controls to have Plasmodium falciparum parasitaemia (S: 176/283 person visits; C: 86/262; P < 0.0001). Parasitaemia was more likely to be associated with febrile symptoms in splenectomized individuals (S: 104/176, 59%; C: 24/86, 28%; P < 0.0001). There were three deaths (two non-malarial, one unexplained) among splenectomized subjects and none in the control group. Parasite densities reached significantly higher levels, and mature parasite stages were more often seen in the peripheral blood, in asplenic individuals. In a partially immune population, asplenic individuals are at increased risk of malarial infections and illness. In a larger group without the benefit of regular review and prompt therapy, there may be an increased risk of life-threatening malaria. Splenectomy should be avoided when possible in an area with endemic transmission of P. falciparum.     

Rift Valley fever virus vaccine trial: study of neutralizing antibody response in humans

The serological response to immunization with a formalin inactivated Rift Valley fever (RVF) vaccine was studied in 963 Swedish UN soldiers serving in the Sinai peninsula. Antibody titres were determined with a plaque reduction neutralization test (PRNT). Attempts were made to give all soldiers three injections (1 ml s.c. days 0, 7-10 and 28-30) but 128 soldiers received only two injections. In a group of 51 fully vaccinated individuals, repeated blood samples were collected. Fifty of the vaccinees seroconverted. Serum collected six weeks after the first vaccination revealed the highest antibody titres. The geometric mean titre then decreased rapidly during the following two weeks. Six months after vaccination sera were collected from 433 vaccinees who had received three injections and 379 had antibodies detectable by PRNT (88% PRNT greater than or equal to 10). The corresponding figures one and two years after vaccination were 223 seropositives out of 255 (91% PRNT greater than or equal to 10) and 91 out of 123 (74% PRNT greater than or equal to 10), respectively. Multiple stepwise regression showed that three injections gave a better antibody response than two injections. This analysis also showed that the magnitude of the antibody response was reduced with increasing age. Slight, local and general side effects were reported in 6% of the vaccinees and these reactions occurred in individuals with relatively higher antibody response.