Levels Of Rabies Virus Neutralizing Antibodies Research Articles

A phase 2b, Randomized, double blinded comparison of the safety and efficacy of the monoclonal antibody mixture SYN023 and human rabies immune globulin in patients exposed to rabies

BackgroundSYN023 is an anti-rabies monoclonal antibody mixture administered as part of post-exposure prophylaxis regimens. The rabies virus neutralizing antibody (RVNA) concentration generally accepted as an adequate immune response to vaccination is ≥ 0.5 IU/mL. MethodsWithin 54 h of potential rabies exposure, 448 patients in two risk substrata of WHO Category III exposure were randomized to receive either 0.3 mg/kg SYN023 or 0.133 mL/kg human rabies immunoglobulin (HRIG) injected in and around the wound site(s) plus a course of rabies vaccination. Patients were followed for safety and absence of rabies for ≥ 365 days. ResultsGMT RVNA was higher with SYN023 throughout the 2-week post-treatment period. In the primary analysis group (n = 368), 99.4 % of SYN023 recipients versus 4.5 % of HRIG recipients had protective RVNA levels on Day 4. On Day 8, 98.1 % SYN023 versus 12.2 % HRIG recipients were protected. The SYN023:HRIG ratio of geometric mean titer of RVNA (RVNA GMTs) on Day 8 (19.42) exceeded the 10 % superiority margin (P < 0.0001) indicating higher Day 8 RVNA with SYN023. On Day 99, the SYN023:HRIG RVNA GMT ratio (0.66) was below the non-inferiority margin of 20 % (P = 0.9485) suggesting some moderation of vaccine immune response by SYN023 relative to HRIG. The ratio of percent SYN023:HRIG recipients achieving RVNA ≥ 0.5 IU/mL on Day 99 (0.98) met the non-inferiority margin of 20 % (P = 0.013) indicating anti-rabies immune response with SYN023 was non-inferior to HRIG despite this effect. There were no probable/confirmed rabies cases in any patient. Study regimens were well tolerated. ConclusionsSYN023 provided higher RVNA than HRIG soon after rabies exposure. By Day 99 post-treatment, GM RVNA with SYN023 was lower than HRIG, however, the percent of SYN023 recipients with a protective response was not inferior at this time point. No rabies cases were reported in the study. The SYN023 safety profile was acceptable.ClinicalTrials.gov ID: NCT03961555.

Comparative Evaluation of Intradermal vis-à-vis Intramuscular Pre-Exposure Prophylactic Vaccination against Rabies in Cattle.

Rabies is a progressively fatal viral disease affecting a wide variety of warm-blooded animals and human beings. With cattle being major part of Indian livestock population, rabies can result in significant financial losses. Immunization of livestock vulnerable to exposure is the best way to control rabies. The present study was undertaken to investigate the efficacy of a rabies pre-exposure prophylactic vaccine administered through different routes and to sequentially monitor the levels of rabies virus-neutralizing antibody (RVNA) titers in cattle. Thirty cattle were divided into five groups of six animals each. Group I and III animals were immunized with 1 mL and 0.2 mL of rabies vaccine through intramuscular (IM) and intradermal (ID) routes, respectively, on day 0, with a booster dose on day 21; Group II and IV animals were immunized with 1 mL and 0.2 mL of rabies vaccine, respectively, without the booster dose; unvaccinated animals served as a control (Group V). Serum samples were collected on days 0, 14, 28, and 90 to estimate RVNA titers using the rapid fluorescent focus inhibition test (RFFIT). The titers were above an adequate level (≥0.5 IU/mL) on day 14 and maintained up to 90 days in all animals administered the rabies vaccine through the IM and ID route with or without a booster dose. The study indicated that both routes of vaccination are safe and effective in providing protection against rabies. Hence, both routes can be considered for pre-exposure prophylaxis. However, the ID route proved to be more economical due to its dose-sparing effect.

Safety and efficacy of rabies immunoglobulin in pediatric patients with suspected exposure

ABSTRACT Rabies is a deadly viral zoonosis with global disease burden. Following exposure to a rabid animal, post-exposure prophylaxis (PEP) is the standard of care for unvaccinated persons. Despite the large proportion of pediatric cases, limited safety and efficacy data exist for use in pediatric patients. We report the safety, efficacy, and immunogenicity of a phase 4, prospective, 2-center, open-label, single-arm clinical trial evaluating human rabies immunoglobulin (HRIG150; KEDRAB 150 IU/mL) as part of PEP in patients (aged <17) with suspected or confirmed rabies exposure, where PEP was indicated. Thirty participants received 20 IU/kg HRIG150 infiltrated into the detectable wound site(s), with any remainder injected intramuscularly, concomitantly with the first of a 4-dose series (days 0, 3, 7, and 14) of rabies vaccine. Rabies virus neutralizing antibody (RVNA) titers and tolerability were assessed on day 14 following administration. Participant safety was monitored for 84 days. No serious adverse events, rabies infections, or deaths were recorded. Twenty-one participants (70.0%) experienced a total of 57 treatment-emergent adverse events (TEAEs) within 14 days following administration. Twelve participants (40.0%) experienced a total of 13 adverse events deemed treatment related. All TEAEs were mild in severity. On day 14, 28 participants (93.3%) had RVNA levels of ≥0.5 IU/mL (mean±standard deviation: 18.89 ± 31.61). These results demonstrate that HRIG150 is well tolerated and effective in pediatric patients as a component of PEP. To the authors’ knowledge, this study is the first to establish pediatric safety and efficacy of HRIG in the US.

Serological Responses of Raccoons and Striped Skunks to Ontario Rabies Vaccine Bait in West Virginia during 2012-2016.

Since the 1990s, oral rabies vaccination (ORV) has been used successfully to halt the westward spread of the raccoon rabies virus (RV) variant from the eastern continental USA. Elimination of raccoon RV from the eastern USA has proven challenging across targeted raccoon (Procyon lotor) and striped skunk (Mephitis mephitis) populations impacted by raccoon RV. Field trial evaluations of the Ontario Rabies Vaccine Bait (ONRAB) were initiated to expand ORV products available to meet the rabies management goal of raccoon RV elimination. This study describes the continuation of a 2011 trial in West Virginia. Our objective was to evaluate raccoon and skunk response to ORV occurring in West Virginia for an additional two years (2012–2013) at 75 baits/km2 followed by three years (2014–2016) of evaluation at 300 baits/km2. We measured the change in rabies virus-neutralizing antibody (RVNA) seroprevalence in targeted wildlife populations by comparing levels pre- and post-ORV during each year of study. The increase in bait density from 75/km2 to 300/km2 corresponded to an increase in average post-ORV seroprevalence for raccoon and skunk populations. Raccoon population RVNA levels increased from 53% (300/565, 95% CI: 50–57%) to 82.0% (596/727, 95% CI: 79–85%) during this study, and skunk population RVNA levels increased from 11% (8/72, 95% CI: 6–20%) to 39% (51/130, 95% CI: 31–48%). The RVNA seroprevalence pre-ORV demonstrated an increasing trend across study years for both bait densities and species, indicating that multiple years of ORV may be necessary to achieve and maintain RVNA seroprevalence in target wildlife populations for the control and elimination of raccoon RV in the eastern USA.

Safety, pharmacokinetics and pharmacodynamics of SYN023 alone or in combination with a rabies vaccine: An open, parallel, single dose, phase 1 bridging study in healthy Chinese subjects

BackgroundSYN023, a mixture of two anti-rabies humanized monoclonal antibodies (mAbs), namely, CTB011 and CTB012, has been developed as a safe and cost-effective alternative to RIG in the use of postexposure prophylaxis (PEP) after rabies exposure. MethodsThis phase I bridging study was designed to compare the pharmacokinetics (PK) of SYN023 (0.3 mg/kg) alone (cohort A, n = 8) or in combination with a rabies vaccine (cohort B, n = 24) in healthy Chinese subjects with those in American subjects to guide the design of further clinical trials. Their safety was assessed for the development of adverse events (AEs) by laboratory examinations. The levels of serum CTB011, CTB012 and anti-drug antibodies (ADAs) were measured longitudinally for 85 days. Serum rabies virus neutralizing antibody (RVNA) levels were measured as a pharmacodynamic (PD) surrogate. ResultsSome subjects with injectable SYN023 at 0.3 mg/kg developed temporary AEs and adverse drug reactions (ADRs) of Grade 1 or 2, particularly in cohort B, probably due to vaccine coadministration. SYN023 injection displayed a typical and reliable PK, similar to that of human IgGs. Following injection with SYN023, Chinese subjects had slower absorption with higher exposures for CTB011 but lower exposures for CTB012 than American subjects. The SYN023 recipients achieved protective levels of RVNA (≥0.5 IU/mL) on day 3 post injection. A few subjects developed temporary ADA, which did not affect the safety and PK/PD of SYN023 in Chinese subjects. ConclusionSYN023 at 0.3 mg/kg is relatively saf e and effective and can be selected for further clinical trials in Chinese subjects. The clinical trial registration number is CTR20190281. http://www.chinadrugtrials.org.cn/eap/clinicaltrials.searchlist?a=a®_no=CTR20190281.

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose (Herpestes Auropunctatus) Sera for Use as an Oral Rabies Vaccination Biological Marker.

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases reported annually. The control of RABV circulation in wildlife reservoirs is typically accomplished by a strategy of oral rabies vaccination (ORV). Currently no wildlife ORV program exists in Puerto Rico. Research into oral rabies vaccines and various bait types for mongooses has been conducted with promising results. Monitoring the success of ORV relies on estimating bait uptake by target species, which typically involves evaluating a change in RABV neutralizing antibodies (RVNA) post vaccination. This strategy may be difficult to interpret in areas with an active wildlife ORV program or in areas where RABV is enzootic and background levels of RVNA are present in reservoir species. In such situations, a biomarker incorporated with the vaccine or the bait matrix may be useful. We offered 16 captive mongooses placebo ORV baits containing ethyl-iophenoxic acid (et-IPA) in concentrations of 0.4% and 1% inside the bait and 0.14% in the external bait matrix. We also offered 12 captive mongooses ORV baits containing methyl-iophenoxic acid (me-IPA) in concentrations of 0.035%, 0.07% and 0.14% in the external bait matrix. We collected a serum sample prior to bait offering and then weekly for up to eight weeks post offering. We extracted Iophenoxic acids from sera into acetonitrile and quantified using liquid chromatography/mass spectrometry. We analyzed sera for et-IPA or me-IPA by liquid chromatography-mass spectrometry. We found adequate marking ability for at least eight and four weeks for et- and me-IPA, respectively. Both IPA derivatives could be suitable for field evaluation of ORV bait uptake in mongooses. Due to the longevity of the marker in mongoose sera, care must be taken to not confound results by using the same IPA derivative during consecutive evaluations.

Timing of Intradermal Rabies Pre-exposure Prophylaxis Injections: Immunological Effect on Vaccination Response.

Rabies remains a global threat, with annually over 59,000 deaths. Intradermal (ID) pre-exposure prophylaxis (PrEP) is very efficient and reduces the need for rabies immunoglobulins. Not much is known about factors that influence the immune response to ID administered rabies vaccine. The aim of this study is to determine if variations in timing of vaccine administration and serology determination, age and gender have an influence on the levels of rabies virus neutralizing antibody (RVNA) after ID rabies vaccination. This is a retrospective study based on electronic health record vaccination data of Belgian military personnel who received ID rabies PrEP with a three injections regimen during the period 2014-2017. Serology was determined by using the RFFIT method. Fischer's exacts tests were used to evaluate the effect of each independent variable on RVNA levels. In this study, 2,112 subjects were included. All but one seroconverted with a RVNA level ≥0.5 IU/mL. About 48% of subjects developed an antibody titer of >10 IU/mL, 36% had antibody levels 3-10 IU/mL and 16% had an antibody level 0.5-2.99 IU/mL. Statistically significant (p = 0.0018) higher RVNA levels are observed in the groups that received vaccination doses later as planned. Timing of serology determination also influenced RVNA levels significantly (p = 0.000). Antibody levels were significantly higher in females than in males (p = 0.000). Age did influence RVNA levels significantly (p = 0.022). Timing of vaccine dose administration, timing of serology testing, sex and age do significantly influence the humoral B-cell response to ID administered rabies vaccine.

Enhanced Immune Response to Rabies Viruses by the Use of a Liposome Adjuvant in Vaccines.

Essen regimen, the Thai Red Cross two-site ID regimen, Zagreb schedule, and the eight-site ID regimen are the standard rabies vaccines recommended by the World Health Organization (WHO). In this study, a liposomal rabies vaccine (LipoRV) was developed, which was found to facilitate the production of rabies virus neutralizing antibody (RVNA) in BALB/c mice. Liposome solution was prepared with hydrogenated soya phosphatide and cholesterol. LipoRV composed of liposome solution and inactivated rabies vaccine (IRV). The immune responses were compared between the mice treated with either LipoRV or IRV. Higher levels of interleukin-2 (p < 0.05), interferon-γ (p < 0.01), and natural killer cell activity (p < 0.05) were observed in the mice immunized with LipoRV than those with IRV. The potency of LipoRV was significantly higher than that IRV (p < 0.05). In addition, three injections of LipoRV on days 0, 3, and 14 could elicit similar RVNA levels as the five shots of IRV. Our data also showed a higher survival rate in mice treated with three shots of LipoRV (56.2%) than five shots of IRV (40.6%). In conclusion, liposome enhances the immune response of mice to rabies vaccine and could be applied as a potential immunopotentiator.

Generation of Recombinant Rabies Virus CVS-11 Expressing eGFP Applied to the Rapid Virus Neutralization Test

The determination of levels of rabies virus-neutralizing antibody (VNA) provides the foundation for the quantitative evaluation of immunity effects. The traditional fluorescent antibody virus neutralization test (FAVN) using a challenge virus standard (CVS)-11 strain as a detection antigen and staining infected cells with a fluorescein isothiocyanate (FITC)-labeled monoclonal antibody, is expensive and high-quality reagents are often difficult to obtain in developing countries. Indeed, it is essential to establish a rapid, economical, and specific rabies virus neutralization test (VNT). Here, we describe a recombinant virus rCVS-11-eGFP strain that stably expresses enhanced green fluorescent protein (eGFP) based on a reverse genetic system of the CVS-11 strain. Compared to the rCVS-11 strain, the rCVS-11-eGFP strain showed a similar growth property with passaging stability in vitro and pathogenicity in vivo. The rCVS-11-eGFP strain was utilized as a detection antigen to determine the levels of rabies VNAs in 23 human and 29 canine sera; this technique was termed the FAVN-eGFP method. The good reproducibility of FAVN-eGFP was tested with partial serum samples. Neutralization titers obtained from FAVN and FAVN-eGFP were not significantly different. The FAVN-eGFP method allows rapid economical, specific, and high-throughput assessment for the titration of rabies VNAs.

Oral vaccination and protection of red foxes (Vulpes vulpes) against rabies using ONRAB®, an adenovirus-rabies recombinant vaccine

Twenty-seven red foxes (Vulpes vulpes) were each offered a bait containing ONRAB®, a recombinant oral rabies vaccine that uses a human adenovirus vector to express the immunogenic rabies virus glycoprotein; 10 controls received no vaccine baits. Serum samples collected from all foxes before treatment, and each week post-treatment for 16 weeks, were tested for the presence of rabies virus neutralizing antibody (RVNA). In the bait group, a fox was considered a responder to vaccination if serum samples from 3 or more consecutive weeks had RVNA ≥0.5IU/ml. Using this criterion, 79% of adult foxes (11/14) and 46% of juveniles (6/13) responded to vaccination with ONRAB®. Serum RVNA of adults first tested positive (≥0.5IU/ml) between weeks 1 and 3, about 4 weeks earlier than in juveniles. Adults also responded with higher levels of RVNA and these levels were maintained longer. Serum samples from juveniles tested positive for 1–4 consecutive weeks; in adults the range was 2–15 weeks, with almost half of adults maintaining titres above 0.5IU/ml for 9 or more consecutive weeks. Based on the kinetics of the antibody response to ONRAB®, the best time to sample sera of wild adult foxes for evidence of vaccination is 7–11 weeks following bait distribution. Thirty-four foxes (25 ONRAB®, 9 controls) were challenged with vulpine street virus 547 days post-vaccination. All controls developed rabies whereas eight of 13 adult vaccinates (62%) and four of 12 juvenile vaccinates (33%) survived. All foxes classed as non-responders to vaccination developed rabies. Of foxes considered responders to vaccination, 80% of adults (8/10) and 67% of juveniles (4/6) survived challenge. The duration of immunity conferred to foxes would appear adequate for bi-annual and annual bait distribution schedules as vaccinates were challenged 1.5 years post-vaccination.

Humoral immune response to oral rabies vaccination in raccoon kits: Problems and implications

Little is known about the immunogenicity of RABORAL V-RG® (V-RG), an oral rabies vaccine, in raccoon kits (Procyon lotor). The objectives of this study were to characterize the immunogenicity of V-RG in young kits and investigate the potential impact of maternal antibodies on response to vaccination of nursing raccoon kits. Raccoon kits (n=30) were vaccinated at either 3 weeks of age, 7 weeks of age, or assigned as contact controls. Nineteen kits (73%) that were whelped by unvaccinated mothers responded to V-RG exposure (orally or indirect contact) by production of detectable rabies virus neutralizing antibodies (RVNA) while 7 (27%) kits did not respond to V-RG exposure. Four kits were whelped by a mother with high levels of RVNA and all four kits acquired maternal rabies antibodies. At approximately 9 months of age, all kits were inoculated with a killed rabies vaccine, IMRAB3®. The kits which initially responded to V-RG oral vaccination or contact with vaccinated littermates demonstrated a rapid anamnestic response. In contrast, the V-RG non-responders and those with acquired maternal antibodies exhibited a primary immune response to IMRAB3®, where RVNA levels were substantially lower on days 5 and 7 than the levels in the animals with an anamnestic response. These findings suggest that the naïve contact kits and the nonresponsive kits most likely remained susceptible to rabies virus infection whereas the ones demonstrating response to V-RG would not have been susceptible to a rabies virus infection.

Relationship between Virus-Neutralizing Antibody Levels and the Number of Rabies Vaccinations: a Prospective Study of Dogs in Japan

A mass rabies vaccination of dogs has been conducted annually in Japan over the last 60 years. To assess both current levels of rabies virus-neutralizing antibody (VNA) in dogs and the rationale for current vaccination procedures, we used a rapid fluorescent focus inhibition test to determine VNA levels in 756 dogs that had visited animal hospitals in Japan. We found that 51.1% of the dogs that had received 1 rabies vaccination had protective VNA levels (≥0.5 IU/ml) with a geometric mean of 0.61 IU/ml. In contrast, 97.8% of the dogs that had been vaccinated at least twice had protective VNA levels with a geometric mean of 7.86 IU/ml. Furthermore, 97.9-100% of the dogs vaccinated at least twice retained protective VNA levels into the second year after the last vaccination. Although VNA levels in the dogs vaccinated at least twice tended to decline 2 years after the last vaccination, 78.9% retained protective VNA levels. Thus, the current rabies vaccination schedule provides adequate protection, but the registration system and vaccination schedule needs to be improved to ensure that increased numbers of dogs are vaccinated against rabies.

Time interval for booster vaccination following reexposure to rabies in previously vaccinated subjects

Background: In rabies endemic areas, re-exposures to rabies are quite common and the incidence could be up to 15%. The recent guidelines of World Health Organization do not specify the duration of protection provided by previous pre- or post exposure prophylaxis. This often puts the treating physician in a dilemma in such cases of re-exposure. Objective: Study the time interval between primary and booster vaccination in individuals who have taken previously a full course of either pre- or post exposure prophylaxis and are now re-exposed to rabies. Methods: The data obtained through a literature search using Pubmed and advanced Google search along with data from in house clinical trials were used for analysis. Sixty-six vaccine cohorts spanning more than 27 years from 1983 to 2010 from six countries were studied. The duration of protection offered by previous vaccination was assessed by using a surrogate marker of adequate (> 0.5 IU per mL) rabies virus neutralizing antibody levels in the individuals vaccinated either by pre-exposure or post exposure regimens received by intramuscular or intradermal routes. Results: The proportions of 2,795 subjects who had received prior post-exposure immunization and produced rabies virus neutralizing antibody levels of less than 0.5 IU per mL were 0.07% and 0.14% at the end of the first and third month post primary vaccination. All 577 subjects with previous pre-exposure vaccination had antibody responses above 0.5 IU per mL at the end of the first and third month post primary vaccination. Conclusion: We concluded that it may be safe for up to three months after previous pre- or post exposure vaccination to not administer boosters to healthy subjects who have been re-exposed to rabies. Keywords: Booster vaccination, post exposure prophylaxis, pre-exposure vaccination, rabies, re-exposure

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).

Oral vaccination of captive arctic foxes with lyophilized SAG2 rabies vaccine.

Arctic foxes (Alopex lagopus) were immunized with lyophilized SAG2 oral rabies vaccine. The effectiveness of this vaccine was determined by serologic response and survival to challenge by rabies virus isolated from a red fox from Alaska (USA). No vaccine virus was found in saliva 1-72 hr after ingestion. At 2 wk after vaccination, all foxes had seroconverted, with rabies virus neutralizing antibody levels of 0.2-3.1 IU ml(-1). All vaccinated foxes survived to week 17 after challenge, and hippocampus, pons, and cerebellum were free of rabies virus as determined by direct immunofluorescence testing after death. One of four nonvaccinated foxes survived challenge and was free of rabies virus in neural tissue, and no rabies virus neutralizing antibody was detected in blood. Our results suggest that the lyophilized SAG2 oral rabies vaccine could be effective in arctic and subarctic regions, where freezing air and ground temperatures probably would not reduce its immunogenicity.

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

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

Preexposure efficacy of a novel combination DNA and inactivated rabies virus vaccine.

Several strategies are being examined to enhance the potency of DNA rabies vaccine (DRV) so that it can be used for both prophylaxis and postexposure therapy of rabies. In this study, we report a novel combination rabies vaccine (CRV) containing a low dose of cell culture-derived inactivated rabies virus vaccine and DRV. Mice immunized with CRV develop higher levels of rabies virus-neutralizing antibodies (RVNA) than those immunized with DRV and are completely protected against peripheral as well as intracerebral rabies virus challenge. The quantity of inactivated rabies virus vaccine required for enhancing the potency of DRV can be 625-fold lower than that of a standard dose of inactivated rabies virus vaccine. CRV induces higher levels of RVNA than DRV in cattle as well. Thus, we demonstrate for the first time that co-inoculation of DNA vaccine and a low dose of inactivated virus vaccine can be developed into a novel cost-effective vaccination strategy for combating rabies in particular, and infectious diseases in general.

Protection from rabies by a vaccinia virus recombinant containing the rabies virus glycoprotein gene.

Inoculation of rabbits and mice with a vaccinia-rabies glycoprotein recombinant (V-RG) virus resulted in rapid induction of high concentrations of rabies virus-neutralizing antibodies and protection from severe intracerebral challenge with several strains of rabies virus. Protection from virus challenge also was achieved against the rabies-related Duvenhage virus but not against the Mokola virus. Effective immunization by V-RG depended on the expression of a rabies glycoprotein that registered proline rather than leucine as the eighth amino acid from its NH2 terminus (V-RGpro8). A minimum dose required for effective immunization of mice was 10(4) plaque-forming units of V-RGpro8 virus. beta-propiolactone-inactivated preparations of V-RGpro8 virus also induced high levels of rabies virus-neutralizing antibody and protected mice against intracerebral challenge with street rabies virus. V-RGpro8 virus was highly effective in priming mice to generate a secondary rabies virus-specific cytotoxic T-lymphocyte response following culture of lymphocytes with either ERA or PM strains of rabies virus.