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 ≥0.5 IU/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 (≥0.5 IU/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 0.5 IU/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.     

An assessment of ONRAB oral rabies vaccine persistence in free-ranging mammal populations in Ontario,Canada

ONRAB^® is a rabies glycoprotein recombinant human adenovirus type 5 oral vaccine developed for application in baits to control rabies in wildlife populations. Prior to widespread use of ONRAB^®, both the safety and effectiveness of this vaccine required investigation. While previous research has focused on field performance and the persistence and pathogenicity of ONRAB^® in captive animals, we sought to examine persistence and shedding of ONRAB^® in populations of free-ranging target and non-target mammals. We collected oral and rectal swab samples from 84 red foxes, 169 striped skunks, and 116 raccoons during 2007 and 2008 in areas where ONRAB^® vaccine baits were distributed. We also analyzed 930 tissue samples, 135 oral swab and 138 rectal swab samples from 155 non-target small mammals from 10 species captured during 2008 at sites treated with high densities of ONRAB^® vaccine baits. Samples were screened for the presence and quantity of ONRAB^® DNA using quantitative real-time PCR. None of the samples that we analyzed from target and non-target species contained quantities of ONRAB^® greater than 10³EU/mL of ONRAB^® DNA which is a limit that has previously been applied to assess viral shedding. This study builds on similar research and suggests that replication of ONRAB^® in animals is short-lived and the likelihood of horizontal transmission to other organisms is low.     

The safety of ONRAB in select non-target wildlife

ONRAB^® is a recombinant human adenovirus type 5 (HAd5) with the rabies glycoprotein gene incorporated into its genome. ONRAB^® has been used in Canada as an oral rabies vaccine in target wildlife species such as: red fox (Vulpes vulpes), raccoon (Procyon lotor), and striped skunk (Mepthis mephitis). We evaluated the safety of ONRAB^® in non-target wildlife species likely to contact the vaccine baits during oral rabies vaccine campaigns in the United States. We investigated the effects of oral inoculation of high titer ONRAB^®, approximately ten times the dose given to target species, in wood rats (Neotoma spp.), eastern cottontail rabbits (Sylvilagus floridanus), Virginia opossums (Didelphis virginiana), eastern wild turkeys (Meleagris gallopavo silvestri), and fox squirrels (Sciurus niger). We performed real-time polymerase chain reaction (PCR) on fecal swabs, oral swabs, and tissues, including lung, liver, kidney, small intestine, large intestine, and when appropriate nasal turbinates, to detect ONRAB^® DNA from inoculated animals. By seven days post-inoculation, turkeys, opossums, and cottontails had all stopped shedding ONRAB^® DNA. One wood rat and one fox squirrel still had detectable levels of ONRAB^® DNA in fecal swabs 14 days post-inoculation. Real-time PCR analysis of the tissues revealed some ONRAB^® DNA persisting in certain tissues; however, there were no significant gross or histologic lesions associated with ONRAB^® in any of the species studied. Our results suggest that many non-target species are not likely to be impacted by the distribution of ONRAB^® as part of oral rabies vaccination programs in the United States.     

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.     

In vitro and in vivo genetic stability studies of a human adenovirus type 5 recombinant rabies glycoprotein vaccine (ONRAB)

Investigation into the genetic stability of a replication-competent human adenovirus rabies glycoprotein recombinant (ONRAB) developed for use as an oral vaccine for wildlife rabies prevention is of major importance due to the vaccine's intended placement in the environment. Using a collection of murine monoclonal antibodies directed to six distinct antigenic sites on the rabies glycoprotein, preservation of all main immunogenic epitopes of the protein after virus growth in vitro was established. A competition experiment which involved the in vitro passaging of a mixture of ONRAB and wild-type human adenovirus type 5 demonstrated that the two viruses do not exhibit noticeably different fitness levels in this environment. Nucleotide sequencing of the expression cassette of multiple viral clones recovered after 20 serial passages in cell culture and 5 serial passages in cotton rats (Sigmodon hispidus), a species susceptible to human adenovirus infection, indicated no changes in comparison to the original virus. These trials demonstrated the stability of the insert gene of ONRAB during in vivo and in vitro passaging.     

Oral vaccination against mycoplasmal pneumonia of swine using a live Erysipelothrix rhusiopathiae vaccine strain as a vector

Erysipelothrix rhusiopathiae Koganei 65-0.15 strain, the live swine erysipelas vaccine for subcutaneous injection, has been shown to colonize the tonsils of pigs after oral inoculation. We thus evaluated the possible use of the strain as a vector for oral vaccination against mycoplasmal pneumonia of swine. Recombinant E. rhusiopathiae strains expressing the C-terminal domain of the P97 adhesin of Mycoplasma hyopneumoniae were constructed and examined for vaccine efficacy in mice and pigs. Mice subcutaneously inoculated with the recombinant strains were protected from challenge exposure to a virulent E. rhusiopathiae. Administration of milk replacer containing recombinant E. rhusiopathiae expressing the M. hyopneumoniae protein protected pigs from death after exposure to E. rhusiopathiae and significantly reduced the severity of pneumonic lung lesions caused by infection with M. hyopneumoniae.     

Oral vaccination of Atlantic salmon (Salmo salar) against salmonid rickettsial septicaemia

Effective oral immunization systems may be very helpful to the salmon industry, particularly during the seawater growth stages in which vaccination through injection is not possible. During the seawater growing stage, fish become more susceptible to several types of disease, due to the natural decay of vaccine-induced immune responses. In this study, we demonstrate the immune response and efficacy of a new salmonid rickettsial septicaemia (SRS) oral vaccine, developed using MicroMatrix™ Technology. The vaccine, which is administered together with daily feed ration, induces a specific immune response at local and systemic levels. Anti-Piscirickettsia salmonis specific antibodies were detected as soon as 300 degree-days after vaccination. Furthermore, oral vaccination was able to protect fish against a lethal pathogen challenge when administered either as a primary vaccination or as a booster for an injected vaccine. Results show that oral vaccination is an efficacious treatment for the prevention of SRS outbreaks throughout the salmon culture period.     

Induction of partial protection against infection with Toxoplasma gondii genotype II by DNA vaccination with recombinant chimeric tachyzoite antigens

Infection with the obligate intracellular parasite Toxoplasma gondii is a significant source of parasitic infections worldwide. In adults, infections may often lead to severe retinochoroiditis. Infection of the foetus causes abortion or congenital pathology that may lead to neurological complications. Although several strategies have been suggested for making a vaccine, none is currently available. Here, we investigate the protection conferred by DNA vaccination with two constructs, pcEC2 (MIC2-MIC3-SAG1) and pcEC3 (GRA3-GRA7-M2AP), encoding chimeric proteins containing multiple antigenic sequences from T. gondii. After challenge with a T. gondii genotype II, but not a genotype III strain, a significant decrease in cerebral cyst load was found compared to the controls. The immune protection involved a cell-mediated immune response with the synthesis of the cytokines IFN-γ and IL-10. In silico structure analysis and the expression profile of EC2, suggest an association between antigen stability, the degree of protein secondary structure and induction of cellular immune responses. Intracellular protein degradation is an important step in the pathway leading to presentation of antigenic peptides on Major Histocompatibility Complex molecules. We suggest that degradation of this chimeric protein may have contributed to the induction of a cellular immune response via enhanced presentation of antigenic peptides on Major Histocompatibility Complex class I molecules.     

Variable protection after vaccination of broiler chickens against necrotic enteritis using supernatants of different Clostridium perfringens strains

Necrotic enteritis is an economically important disease of chickens caused by Clostridium perfringens. Immunity to necrotic enteritis is not fully characterized yet, but previous reports indicate that immunoprotective potential is present in the secreted component of C. perfringens. This study aimed to compare the vaccine potential of the supernatants of eight chicken strains of C. perfringens differing in origin, level of alpha toxin production and presence of netB gene. The supernatant of only one strain provided full protection, while one other strain provided partial protection against a severe infection challenge. Our results indicate that the protective characteristics of the supernatants are not solely based on the presence of NetB or alpha toxin.     

Oral immunization with Lactococcus lactis-expressing EspB induces protective immune responses against Escherichia coli O157:H7 in a murine model of colonization

Enterohemorrhagic Escherichia coli (EHEC) have been responsible for several outbreaks of hemolytic–uremic syndrome (HUS) worldwide. HUS is the most common cause of acute renal failure in children and results in fatalities as high as 50% in the elderly. Currently, neither a specific treatment nor a vaccine is available for EHEC. Lactococcus lactis is a generally regarded as safe “GRAS” bacterium that offers a valuable platform for oral vaccine delivery. Toward the development of an oral vaccine against EHEC, we have previously constructed a recombinant L. lactis strain expressing the EHEC antigen, EspB in the cytoplasmic compartment. However, oral immunization of mice with this strain induced weak priming of the immune system. This outcome was attributed to the rather low levels of EspB expressed by this recombinant strain. Therefore, in the present study we optimized the expression of EspB in L. lactis by secreting the antigen either under constitutive or nisin-inducible control. Indeed, oral immunization of mice with the EspB-secreting strains successfully induced specific mucosal and systemic antibody responses. These responses were associated with mixed Th1/Th2 cell responses in Peyer's Patches and mesenteric lymph nodes. Moreover, immunized mice exhibited significant protection against E. coli O157:H7 colonization, as indicated by the reduced amount and/or duration of the bacterial fecal shedding. Our results demonstrate the protective potential of EspB as an oral vaccine against EHEC infection. Additionally, the study demonstrates the efficient delivery of recombinant EspB by the “GRAS” bacterium, L. lactis. The safety profile of L. lactis as a vaccine vehicle can particularly be beneficial to children and elderly as high-risk groups for HUS incidence.     

Oral delivery of a DNA vaccine against tuberculosis using operator-repressor titration in a Salmonella enterica vector

Attenuated Salmonella enterica offers a vaccine delivery route that has the benefits of enhanced immunogenicity and oral delivery. The majority of immunization studies have been conducted to deliver recombinant proteins, expressed from a gene that is either chromosomally integrated or carried on a low- or medium-copy number plasmid. There are, however, an increasing number of reports demonstrating the delivery of DNA vaccines, but the high-copy number plasmids that are preferentially used for this application are unstable in Salmonella. Here, we use the Operator-Repressor Titration (ORT) plasmid maintenance system in Salmonella enterica serovar Typhimurium to deliver a high-copy number plasmid expressing the Mycobacterium tuberculosis gene mpt64 to mice. MPT64 expression was detected in phagocytes using immunofluorescence microscopy following Salmonella-mediated delivery of the DNA vaccine. The indicative CD8+ responses measured by antigen-specific IFN-γ were higher from the live bacterial vector than from injected plasmid DNA, and a reduction in the pulmonary bacterial load was seen following an aerogenic challenge. This illustrates the potential of live attenuated Salmonella as oral tuberculosis vaccine vectors.     

Efficacy of Hyalomma scupense (Hd86) antigen against Hyalomma excavatum and H. scupense tick infestations in cattle

The Rhipicephalus microplus recombinant Bm86-based tick vaccines have shown their efficacy for the control of several Hyalomma cattle ticks genera, namely H. dromedarii and H. anatolicum. However, H. scupense species, the most important tick in North Africa has never been studied. Vaccination trials using either a recombinant Bm86-based vaccine or a recombinant Hd86-based vaccine (the Bm86 ortholog in H. scupense) were conducted in cattle against immature and adult H. scupense ticks and adult H. excavatum ticks. The results showed a 59.19% reduction in the number of scupense nymphs engorging on Hd86 vaccinated cattle. However, cattle vaccination with Bm86 or Hd86 did not have an effect on H. scupense or H. excavatum adult ticks infestations. These results showed that Hd86 vaccines are selectively effective against H. scupense immature instars and emphasize on an integrated anti-tick vaccine control in North Africa.     

T-cell-derived cytokines,nitric oxide production by peripheral blood monocytes and seric anti-Leishmania (Leishmania) chagasi IgG subclass patterns following immunization against canine visceral leishmaniasis using Leishvaccine and Leishmune

It is generally accepted that distinct cytokine expression by the cellular immune response plays a critical role during the outcome of experimental as well as natural canine visceral Leishmaniasis (CVL). Despite the fact that immunoprophylaxis of CVL has become an important control strategy and protective immunity has been reported upon immunization with whole as well as purified Leishmania antigens, the cytokine profile of T-cells triggered by anti-CVL vaccines still remain to be determined. Herein, we have developed a cross-sectional analysis of German Shepherd dogs submitted to vaccination protocols with Leishvaccine (n = 6) and Leishmune^® (n = 6). Our data identified distinct immunological profiles elicited by Leishvaccine and Leishmune^®, with the Leishvaccine triggering a mixed, IFN-γ and IL-4, cytokine pattern in addition to high levels of anti-Leishmania IgG1, whereas the Leishmune^® induced an immunological pattern characterized by enhanced levels of IFN-γ, NO and anti-Leishmania chagasi IgG2. It was important to notice that despite the distinct immunological patterns triggered by Leishvaccine and Leishmune^®, the ability of both immunobiologicals to activate T-cell-derived IFN-γ synthesis further suggesting their immunogenic potential against CVL. These findings added support to our hypothesis that both antigenic composition (whole antigen in Leishvaccine versus purified antigen in Leishmune^®) as well as the adjuvant nature (BGC and saponin) used for the vaccine formulation may count for the distinct activation pattern observed.     

Current status and prospects for development of a vaccine against Trichomonas vaginalis infections

Trichomonas vaginalis is a sexually transmitted pathogen with an annual worldwide incidence of over 276 million infections, the highest of all curable and non-viral STI. A large proportion of cases are asymptomatic and under-diagnosed with conventional diagnostic tools. Infection has important maternal and fetal health consequences and can lead to a higher probability of HIV transmission and susceptibility. Lack of affordable accurate diagnostic tests globally and metronidazole resistance hinder T. vaginalis control efforts. Based on data from current vaccination studies in animal models, a human vaccine is achievable to intervene on the substantial incidence of infection.     

Enhancement of protective immune responses induced by Toxoplasma gondii dense granule antigen 7 (GRA7) against toxoplasmosis in mice using a prime-boost vaccination strategy

Effective vaccines against Toxoplasma gondii may contribute to preventing and controlling the spread of toxoplasmosis, which is important for improving outcomes of infections in humans and livestock animals. The dense granule antigen 7 (GRA7) of T. gondii might be an immunodominant antigen for a vaccine candidate. In the present study, a further exploration of its vaccine effect, a heterologous prime-boost vaccination strategy with a recombinant eukaryotic plasmid pEGFP-GRA7 and a recombinant protein GRA7 expressed from a prokaryotic plasmid pET30-GRA7, was performed in BALB/c mice. The data reveal that a DNA prime-protein boost vaccination induces both humoral and cellular immune responses against T. gondii associated with high levels of total IgG, IgG2a isotype and gamma interferon (IFN-γ). Challenge experiments further show that the DNA prime-protein boost vaccination significantly increases survival rate (60%), compared with controls in which all died within 8 days of challenge. Therefore, the DNA prime-protein boost vaccination based on GRA7 might be a promising regimen for further development of an effective vaccine against T. gondii.     

Efficacy of a divalent and a multivalent water-in-oil formulated vaccine against a highly virulent strain of Flavobacterium psychrophilum after intramuscular challenge of rainbow trout (Oncorhynchus mykiss)

Flavobacterium psychrophilum is a well-known pathogen causing significant problems in aquaculture worldwide. In recent years an increasing number of disease outbreaks caused by F. psychrophilum has been reported on juvenile and post smolts of rainbow trout (Oncorhynchus mykiss) in Norway. The current study was performed to assess the efficacy of two autogenous water-in-oil formulated vaccines containing whole cell antigens of F. psychrophilum to induce protective immunity against challenge. The vaccines were formulated either as multivalent (FLAVO AVM6) or divalent (FLAVO IPN) and administered by the intraperitoneal route. Intramuscular challenge with a field strain of F. psychrophilum was carried out 552 day degrees post vaccination, at a time when the FLAVO AVM6 and FLAVO IPN vaccinated groups had significantly higher antibody responses compared to the negative control. Results from the challenge study showed that the multivalent and the divalent vaccines had capacity to induce significant protection, with RPS₆₀ > 87% and RPS_end > 77.5% for both vaccines. The high level of protection seen in the vaccinated groups was also reflected in the reduced ulceration rates observed at the injection site. Combining our results demonstrate that vaccination with FLAVO AVM6 and FLAVO IPN induces responses capable of protecting rainbow trout against infections with F. psychrophilum.     

Protection of cattle against a natural infection of Fasciola hepatica by vaccination with recombinant cathepsin L1 (rFhCL1)

The liver fluke, Fasciola hepatica causes liver fluke disease, or fasciolosis, in ruminants such as cattle and sheep. An effective vaccine against the helminth parasite is essential to reduce our reliance on anthelmintics, particularly in light of frequent reports of resistance to some frontline drugs. In our study, Friesian cattle (13 per group) were vaccinated with recombinant F. hepatica cathepsin L1 protease (rFhCL1) formulated in mineral-oil based adjuvants, Montanide™ ISA 70VG and ISA 206VG. Following vaccination the animals were exposed to fluke-contaminated pastures for 13 weeks. At slaughter, there was a significant reduction in fluke burden of 48.2% in the cattle in both vaccinated groups, relative to the control non-vaccinated group, at p ≤ 0.05. All vaccinated animals showed a sharp rise in total IgG levels to rFhCL1 post-vaccination which was maintained over the course of the 13-week challenge infection and was significantly higher than levels reached in the control group. Arginase levels in the macrophages of vaccinated cattle were significantly lower than those of the control cattle, indicating that the parasite-induced alternative-activation of the macrophages was altered by vaccination. The data demonstrate the potential for recombinant FhCL1 vaccine in controlling fasciolosis in cattle under field conditions.     

Effect of delayed anthrax vaccine dose on Bacillus anthracis protective antigen IgG response and lethal toxin neutralization activity

We describe the Bacillus anthracis protective antigen IgG antibody response and the B. anthracis lethal toxin neutralization activity to a delayed dose of anthrax vaccine adsorbed (AVA, BioThrax^®) using validated assays. 373 individuals received 1, 2, or 3 priming doses, 18–24 months afterward, they received a delayed dose of AVA. Overall, 23.6% of subjects showed detectable anti-PA IgG before the boost, compared to 99.2% (P < 0.0001) 28 days after the boost. Geometric mean anti-PA IgG concentration (GMC) was 1.66 μg/mL before and 887.82 μg/mL after the boost (P < 0.0001). The proportion of individuals with four-fold increase in GMC following the boost ranged from 93.8% to 100%. Robust anti-PA IgG levels and B. anthracis lethal toxin neutralization activity are induced when an AVA dose is delayed as long as two years. These data support continuing with the vaccination schedule when a dose is delayed as long as two years rather than restarting the series.     

Effectiveness of intranasal vaccination against Angiostrongylus costaricensis using a serine/threonine phosphatase 2 A synthetic peptide and recombinant antigens

Intranasal immunization was assayed in C57BL/6 mice against Angiostrongylus costaricensis using a synthetic and a recombinant peptide belonging to the catalytic region of the serine/threonine phosphatase 2 A (PP2A) of the parasite. Immunization was carried out with the synthetic peptide (SP) polymerized either with itself or with the β fraction of the cholera toxin (CTB) and then enclosed in nanocapsules of phosphatidyl choline, cholesterol and Quil A (ISCOM). Another group of mice was immunized with recombinant peptide. Immunization consisted of two intranasal inoculations at two-week intervals, and the challenge with L3 larvae was made one month after the last vaccination.