Single-dose Immunization Research Articles

The immunogenic characteristics associated with multivalent display of Vi polysaccharide antigen using biodegradable polymer particles

Polysaccharides in their great majority are thymus-independent (TI) antigens. Anti-polysaccharide antibody responses are generally weak and characterized by lack of memory, isotype restriction and delayed ontogeny. We report here the generation of protective memory antibody response by multivalent display of polysaccharide antigens on biodegradable polymeric particles. Single dose immunization using polylactide (PLA) polymer particles entrapping Vi capsular polysaccharide antigen from Salmonella typhi promoted isotype switching and induced polysaccharide-specific memory antibody response in experimental animals. PLA nanoparticles as well as microparticles entrapping Vi polysaccharides elicited high IgG titer in comparison to soluble Vi immunization. Immunizations with particles co-entrapping both Vi polysaccharide and tetanus toxoid did not improve the anti-polysaccharide antibody responses. Lower antibody response from co-entrapped formulation was mostly due to inhibition of particle phagocytosis by the macrophages. Immunization using polylactide particles entrapping only Vi polysaccharide with higher density on surface elicited highest secondary antibody response as well as promoted isotype switching. The vaccination potential of particle based immunizations was further confirmed by the generation of quick memory antibody responses while challenging the immunized animals with live S. typhi. This approach provides a multivalent display of polysaccharide antigen using polymer particles and elicits protective memory antibody response without conjugation to a carrier protein

A novel human Neonatal Tissue Construct (NTC) models age-specific immune responses to Bacille Calmette-Guérin (BCG) vaccine (166.27)

Abstract Newborns have a distinct immune system that impairs immune responses to many conventional pediatric vaccines rendering them susceptible to preventable infections. Since birth is a reliable point of healthcare contact worldwide, effective single-dose birth immunization could help reduce global morbidity and mortality. However, vaccine development has been largely ad hoc and empiric. Modeling age-specific human immune responses could help predict safety and efficacy of adjuvants and vaccines targeting the newborn. Using human primary endothelial cells, human extracellular matrix, newborn monocytes and 100% autologous intact plasma we assembled the Neonatal Tissue Construct (NTC). In this tri-dimensional micro-vascular interstitium extravasated CD33+ monocytes autonomously differentiated into HLA-DRhigh CD86high and CCR7+ mature Dendritic Cells (DCs) in response to the live mycobacterial BCG vaccine known to be safe and effective at birth, and to induce effector T cell responses. BCG-pulsed NTC-derived DCs induced autologous naïve CD4+ T cell proliferation and activation (CD45RO+, TNF-α), as efficiently as DCs from an Adult Tissue Construct. While newborn and adult DC:T cell co-cultures secreted IL-2 and TNF-α in response to BCG, only newborn cells demonstrated constitutive IL-4 production and a marked impairment in IFN-γ production. In recapitulating neonatal responses to BCG, the NTC demonstrates its potential as a novel platform to study age-specific vaccine responses.

Immunogenicity of a lentiviral-based DNA vaccine driven by the 5′LTR of the naturally attenuated caprine arthritis encephalitis virus (CAEV) in mice and macaques

Increasing the safety and the efficacy of existing HIV vaccines is one of the strategies that could help to promote the development of a vaccine for human use. We developed a HIV DNA vaccine (Δ4-SHIVKU2) that has been shown to induce potent polyfunctional HIV-specific T cell responses following a single dose immunization of mice and macaques. Δ4-SHIVKU2 also induced protection when immunized macaques were challenged with homologous pathogenic viruses. In the present study, our aim was to examine whether a chimeric HIV DNA vaccine (CAL-Δ4-SHIVKU2) whose genome is driven by the LTR of the goat lentivirus, caprine arthritis encephalitis (CAEV) expresses efficiently the vaccine antigens and induces potent immune responses in animal models for HIV vaccine. Data of radioimmunoprecipitation assays clearly show that this chimeric genome drives efficient expression of all HIV antigens in the construct. In addition, evaluation of the p24 Gag protein in the supernatant of HEK-293-T cells transfected in parallel with Δ4-SHIVKU2 and CAL-Δ4-SHIVKU2 showed no difference suggesting that these two LTRs are inducing equally the expression of the viral genes. Immunization of mice and macaques using our single dose immunization regimen resulted in induction of similar IFN-γ ELISPOT responses in Δ4-SHIVKU2- and CAL-Δ4-SHIVKU2-treated mice. Similar profiles of T cell responses were also detected both in mice and macaques when multiparametric flow cytometry analyses were performed. Since CAEV LTR is not dependent of Tat to drive viral gene expression and is not functional for integration with HIV integrase, this new vector increases the safety and efficacy of our vaccine vectors and vaccination strategy.

Single-Dose Immunization with Virus Replicon Particles Confers Rapid Robust Protection against Rift Valley Fever Virus Challenge

Rift Valley fever virus (RVFV) causes outbreaks of severe disease in people and livestock throughout Africa and the Arabian Peninsula. The potential for RVFV introduction outside the area of endemicity highlights the need for fast-acting, safe, and efficacious vaccines. Here, we demonstrate a robust system for the reverse genetics generation of a RVF virus replicon particle (VRP(RVF)) vaccine candidate. Using a mouse model, we show that VRP(RVF) immunization provides the optimal balance of safety and single-dose robust efficacy. VRP(RVF) can actively synthesize viral RNA and proteins but lacks structural glycoprotein genes, preventing spread within immunized individuals and reducing the risk of vaccine-induced pathogenicity. VRP(RVF) proved to be completely safe following intracranial inoculation of suckling mice, a stringent test of vaccine safety. Single-dose subcutaneous immunization with VRP(RVF), although it is highly attenuated, completely protected mice against a virulent RVFV challenge dose which was 100,000-fold greater than the 50% lethal dose (LD(50)). Robust protection from lethal challenge was observed by 24 h postvaccination, with 100% protection induced in as little as 96 h. We show that a single subcutaneous VRP(RVF) immunization initiated a systemic antiviral state followed by an enhanced adaptive response. These data contrast sharply with the much-reduced survivability and immune responses observed among animals immunized with nonreplicating viral particles, indicating that replication, even if confined to the initially infected cells, contributes substantially to protective efficacy at early and late time points postimmunization. These data demonstrate that replicon vaccines successfully bridge the gap between safety and efficacy and provide insights into the kinetics of antiviral protection from RVFV infection.

Nanovaccines : nanocarriers for antigen delivery

Vaccination has become one of the most important health interventions of our times, revolutionizing health care, and improving the quality of life and life expectancy of millions all over the world. In spite of this, vaccine research remains a vast field for innovation and improvement. Indeed, the shift towards the use of sub-unit antigens, much safer but less immunogenic, and the recognized need to facilitate the access to vaccines in the global framework is currently stimulating the search for safe and efficient adjuvants and delivery technologies. Within this context, nanocarriers have gained particular attention over the last years and appear as one of the most promising strategies for antigen delivery. A number of biomaterials and technologies can be used to design nanovaccines that fulfill the requirements of new vaccination approaches, such as single-dose and transmucosal immunization, critical for achieving a widespread coverage while reducing the overall costs in relation to traditional forms of vaccination. Here we present an overview of the current state of nanocarriers for antigen delivery, developed with the perspective of contributing to the global vaccination goal.

Rectal single dose immunization of mice with Escherichia coli O157:H7 bacterial ghosts induces efficient humoral and cellular immune responses and protects against the lethal heterologous challenge

SummaryBacterial ghosts (BGs) have been applied through oral, aerogenic, intraocular or intranasal routes for mucosal immunization using a wide range of experimental animals. All these applications required a booster after primary immunization to achieve protective immunity against the lethal challenge. Here we report for the first time that a single rectal dose of BGs produced from enterohaemorrhagic Escherichia coli (EHEC) O157:H7 fully protects mice against a 50% lethal challenge with a heterologous EHEC strain given at day 55. BGs from EHEC O157:H7 were prepared by a combination of protein E‐mediated cell lysis and expression of staphylococcal nuclease A guaranteeing the complete degradation of pathogen residual DNA. The lack of genetic material in the EHEC BGs vaccine abolished any potential hazard for horizontal gene transfer of plasmid encoded antibiotic resistance genes or pathogenic islands to the recipient's gut flora. Single rectal immunization using EHEC O157:H7 BGs without any addition of adjuvant significantly stimulated efficient humoral and cellular immune responses, and was equally protective as two immunizations, which indicates the possibility to develop a novel efficacious single dose mucosal EHEC O157:H7 BGs vaccine using a simplified immunization regimen.

Protection from Experimental Cerebral Malaria with a Single Dose of Radiation-Attenuated, Blood-Stage Plasmodium berghei Parasites

BackgroundWhole malaria parasites are highly effective in inducing immunity against malaria. Due to the limited success of subunit based vaccines in clinical studies, there has been a renewed interest in whole parasite-based malaria vaccines. Apart from attenuated sporozoites, there have also been efforts to use live asexual stage parasites as vaccine immunogens.Methodology and ResultsWe used radiation exposure to attenuate the highly virulent asexual blood stages of the murine malaria parasite P. berghei to a non-replicable, avirulent form. We tested the ability of the attenuated blood stage parasites to induce immunity to parasitemia and the symptoms of severe malaria disease. Depending on the mouse genetic background, a single high dose immunization without adjuvant protected mice from parasitemia and severe disease (CD1 mice) or from experimental cerebral malaria (ECM) (C57BL/6 mice). A low dose immunization did not protect against parasitemia or severe disease in either model after one or two immunizations. The protection from ECM was associated with a parasite specific antibody response and also with a lower level of splenic parasite-specific IFN-γ production, which is a mediator of ECM pathology in C57BL/6 mice. Surprisingly, there was no difference in the sequestration of CD8+ T cells and CD45+ CD11b+ macrophages in the brains of immunized, ECM-protected mice.ConclusionsThis report further demonstrates the effectiveness of a whole parasite blood-stage vaccine in inducing immunity to malaria and explicitly demonstrates its effectiveness against ECM, the most pathogenic consequence of malaria infection. This experimental model will be important to explore the formulation of whole parasite blood-stage vaccines against malaria and to investigate the immune mechanisms that mediate protection against parasitemia and cerebral malaria.

Changes of immune parameters in periphery blood of <I>Spinibarbus sinensis</I> Bleeker immunized with formalin-killed <I>Aeromonas sobria</I>

Spinibarbus sinensis Bleeker in the Yangtze River and its tributaries is of great economical value.However,it suffers from serious diseases,particularly a bacterial disease named muscle ulcerationin intensive aquaculture.In order to effectively control the disease of S.sinensis,it is necessary to clarify the changes of immune parameters of periphery blood in S.sinensis immunized with a kind of dead bacteria.One hundred and eighty healthy fish(S.sinensis) with an average body weight of(100±25) g were divided randomly into immunized and control groups.Each individual was intraperitoneally injected with 0.2 mL 1.0×108 mL formalin-killed Aeromonas sobria in immunized group,and with 0.2 mL 0.65% sterile saline solution in control group.Then,approximate 2 mL of blood was taken from each fish caudal vein on Day 0,1,2,4,7,14,21,28 and 35 post injection to study the changes of immune parameters,such as haematocyte number,differential leucocyte count(DLC),serum antibody titer,phagocytic activity of phagocytes and serum protein content.The results were as follows:the number of erythrocytes and leucocytes in peripheral blood of immunized S.sinensis increased during the first 4 days post injection and peaked on 4th day,then,gradually decreased to the control level(Fig.1-a 1-b).The differential leucocyte count(DLC) of the immunized group increased and was significantly(or very significantly) higher than that of control group for monocytes and neutrophils from day 4 to day 7(Fig.2-a 2-b),and for lymphocytes from the 14th day to 28th day(Fig.2-c),but lower than that of control group for thrombocytes on day 4 and 21(Fig.2-d),and was the same level as that of control group for the four kinds of cells at the other sampling times.The phagocytic percentage(PP) and phagocitic index(PI) of phagocytes in immunized S.sinensis were significantly higher than those of the non-immunized fish between day 2 and day 7,and of the maximum value on day 4,but were not significantly different from those of the non-immunized S.sinensis at the other sampling times(Fig.3-a 3-b).The serum antibody titers gradually increased in immunized S.sinensis during the first 21 days post injection and reached the greatest value on day 21,then,gradually dropped off,but always kept the low level of 1:4-1:5 in the non-immunized fish(Tab.1).The serum globulin content gradually increased in the immunized fish from day 4 to day 21,peaked and significantly higher than that of the non-immunized fish on day 21(Tab.2).The serum globulin changes resulted in the same change model of serum total protein,which was not of significant difference between immunized and control groups(Tab.2).However,the serum albumin quantity was not significantly different between the immunized and control groups,and fluctuated a little in the two groups(Tab.2).Relative percent survival was 65.21% for the immunized S.sinensis after challenge by means of injection with 0.2 mL live A.sobria at a concentration of 5.0×107 mL for every individual(Tab.3).This indicated that the formalin-killed Aeromonas sobria(F-AS) stimulated S.sinensis to produce a significantly protective immunity against the pathogen bacteria infection.All the results stated above suggested that the vaccine(F-AS) made S.sinensis enhance the non-specific cell immunity,such as monocytes,neutrophils and erythrocytes,during the early period(from day 1 to day 7),and the specific cellular and humoral immunity,such as lymphocytes and antibodies,during the later period against the bacterial pathogen infection post single dose immunization,and the relative percent survival reached 65.21% for the immunized S.sinensis after challenge with live A.sobria.

Single-Dose Mucosal Immunization with a Candidate Universal Influenza Vaccine Provides Rapid Protection from Virulent H5N1, H3N2 and H1N1 Viruses

BackgroundThe sudden emergence of novel influenza viruses is a global public health concern. Conventional influenza vaccines targeting the highly variable surface glycoproteins hemagglutinin and neuraminidase must antigenically match the emerging strain to be effective. In contrast, “universal” vaccines targeting conserved viral components could be used regardless of viral strain or subtype. Previous approaches to universal vaccination have required protracted multi-dose immunizations. Here we evaluate a single dose universal vaccine strategy using recombinant adenoviruses (rAd) expressing the conserved influenza virus antigens matrix 2 and nucleoprotein.Methodology/Principal FindingsIn BALB/c mice, administration of rAd via the intranasal route was superior to intramuscular immunization for induction of mucosal responses and for protection against highly virulent H1N1, H3N2, or H5N1 influenza virus challenge. Mucosally vaccinated mice not only survived, but had little morbidity and reduced lung virus titers. Protection was observed as early as 2 weeks post-immunization, and lasted at least 10 months, as did antibodies and lung T cells with activated phenotypes. Virus-specific IgA correlated with but was not essential for protection, as demonstrated in studies with IgA-deficient animals.Conclusion/SignificanceMucosal administration of NP and M2-expressing rAd vectors provided rapid and lasting protection from influenza viruses in a subtype-independent manner. Such vaccines could be used in the interval between emergence of a new virus strain and availability of strain-matched vaccines against it. This strikingly effective single-dose vaccination thus represents a candidate off-the-shelf vaccine for emergency use during an influenza pandemic.

Long-term immunity and immune response to a booster dose following vaccination with the inactivated Japanese encephalitis vaccine IXIARO®, IC51

IC51 (IXIARO®, JESPECT®) is a recently approved prophylactic Japanese encephalitis virus vaccine with a two-vaccine primary immunization regimen. In this phase 3 trial, after primary immunization with a Day 0/28 dose schedule, seroprotection rates were 83%, 58% and 48% at Month 6, Month 12 and Month 24, respectively. A booster dose at Month 11 and/or Month 23 in subjects with neutralizing antibody titers below the limit of detection (defined as a serum dilution giving a 50% reduction of plaque counts in a plaque reduction neutralization test [PRNT50]<1:10) led to 100% seroconversion. After a single-dose immunization (incomplete primary immunization), only 9% of subjects were seroprotected at Month 6; however, a booster dose at Month 11 led to seroconversion in 99% of subjects. Hence, subjects with incomplete primary immunization can complete their schedule within at least 11 months.

A highly attenuated recombinant human respiratory syncytial virus lacking the G protein induces long-lasting protection in cotton rats

BackgroundRespiratory syncytial virus (RSV) is a primary cause of serious lower respiratory tract illness for which there is still no safe and effective vaccine available. Using reverse genetics, recombinant (r)RSV and an rRSV lacking the G gene (ΔG) were constructed based on a clinical RSV isolate (strain 98-25147-X).ResultsGrowth of both recombinant viruses was equivalent to that of wild type virus in Vero cells, but was reduced in human epithelial cells like Hep-2. Replication in cotton rat lungs could not be detected for ΔG, while rRSV was 100-fold attenuated compared to wild type virus. Upon single dose intranasal administration in cotton rats, both recombinant viruses developed high levels of neutralizing antibodies and conferred comparable long-lasting protection against RSV challenge; protection against replication in the lungs lasted at least 147 days and protection against pulmonary inflammation lasted at least 75 days.ConclusionCollectively, the data indicate that a single dose immunization with the highly attenuated ΔG as well as the attenuated rRSV conferred long term protection in the cotton rat against subsequent RSV challenge, without inducing vaccine enhanced pathology. Since ΔG is not likely to revert to a less attenuated phenotype, we plan to evaluate this deletion mutant further and to investigate its potential as a vaccine candidate against RSV infection.

A candidate H1N1 pandemic influenza vaccine elicits protective immunity in mice.

BackgroundIn 2009 a new pandemic disease appeared and spread globally. The recent emergence of the pandemic influenza virus H1N1 first isolated in Mexico and USA raised concerns about vaccine availability. We here report our development of an adenovirus-based influenza H1N1 vaccine tested for immunogenicity and efficacy to confer protection in animal model.MethodsWe generated two adenovirus(Ad5)-based influenza vaccine candidates encoding the wildtype or a codon-optimized hemagglutinin antigen (HA) from the recently emerged swine influenza isolate A/California/04/2009 (H1N1)pdm. After verification of antigen expression, immunogenicity of the vaccine candidates were tested in a mouse model using dose escalations for subcutaneous immunization. Sera of immunized animals were tested in microneutalization and hemagglutination inhibition assays for the presence of HA-specific antibodies. HA-specific T-cells were measured in IFNγ Elispot assays. The efficiency of the influenza vaccine candidates were evaluated in a challenge model by measuring viral titer in lung and nasal turbinate 3 days after inoculation of a homologous H1N1 virus.Conclusions/SignificanceA single immunization resulted in robust cellular and humoral immune response. Remarkably, the intensity of the immune response was substantially enhanced with codon-optimized antigen, indicating the benefit of manipulating the genetic code of HA antigens in the context of recombinant influenza vaccine design. These results highlight the value of advanced technologies in vaccine development and deployment in response to infections with pandemic potential. Our study emphasizes the potential of an adenoviral-based influenza vaccine platform with the benefits of speed of manufacture and efficacy of a single dose immunization.

Intragastric immunization of mice with enterohemorrhagicEscherichia coliO157:H7 bacterial ghosts reduces mortality and shedding and induces a Th2-type dominated mixed immune response

A bacterial ghost (BG)-based vaccine was developed against enterohemorrhagic Escherichia coli O157:H7 infection. The aim of this study was to evaluate the protective effect of E. coli O157:H7 BGs in a mouse model and to reveal the mechanism of the immune response. Booster immunization provided a higher protection rate (84%) than single-dose immunization (56%). Intragastric immunization of E. coli O157:H7 BGs induced both humoral and cellular immune responses. The proliferative response of CD4+ T cells was mediated by the antigen-presenting cells. The humoral immune response dominated the immune response, while the cellular immune response developed later. Inflammatory reaction was balanced by the mixed Th1/Th2 immune response. The immune sera anti-adhesion effect was confirmed by the inhibition effect, which could inhibit >90% of the adhesion of E. coli O157:H7 to Hep-2 target cells in vitro. Antibody titer specific for intimin, a molecule important for adhesion of E. coli O157:H7 to target cells, correlated with specific immunoglobulin A or G antibody titer. Therefore, it might be feasible to clinically test BG vaccines in the future.

Humoral immune responses and protective efficacy of sequential B- and T-cell epitopes of V antigen of Yersinia pestis by intranasal immunization in microparticles

Capsular F1 and secretory V antigen are the putative vaccine candidates for plague, caused by Yersinia pestis. Contemplating this, we studied the immunogenicity and protective efficacy of collinearly synthesized B- and T-cell epitopes (B-T constructs) of V antigen entrapped in poly (DL-lactide-co-glycolide) microparticles immunized intranasally using single dose immunization schedule in outbred, H-2(b) and H-2(d) mice. High antibody levels were observed in terms of IgG, IgA and SIgA peak titers in sera and mucosal washes to different B-T constructs. The constructs ai, bi and fi especially showed high peak antibody titers ranging from 51,200 to 204,000, which were maintained till day 120 post immunization. IgG/IgA Specific activity in sera and washes correlated well with the peak antibody titers. Moreover, all the B-T constructs showed mixed IgG1 and IgG2a/2b response, variable immunoreactivity as well as memory response with V antigen. B-T constructs, viz ai, ak, bi, fi, di and ik showed comparatively high isotype levels. These constructs showed high immunoreactivity, and good recall response with V antigen. Finally, in vivo protective study in BALB/c mice demonstrated the protective efficacy of three B-T constructs (ai, bi and fi) against lethal doses of Yersinia pestis till day 20 post challenge, while construct 'id' showed partial protection.

The Human Cancer Antigen Mesothelin Is More Efficiently Presented to the Mouse Immune System when Targeted to the DEC‐205/CD205 Receptor on Dendritic Cells

To develop a tumor vaccine directly targeting tumor antigen to dendritic cells in situ, we engineered human mesothelin (MSLN) into an antibody specific for mouse DEC-205, a receptor for antigen presentation. We then characterized both T cell and humoral responses to human MSLN and compared immunizing efficacy of DEC-205-targeted MSLN to nontargeted protein after a single-dose immunization. Targeting human MSLN to DEC-205 receptor induced stronger CD4(+) T-cell responses compared to high doses of mesothelin protein. Approximately 0.5% CD4(+) T cells were primed to produce IFN-gamma, tumor necrosis factor-alpha, and IL-2 via intracellular cytokine staining, and the T cells also could proliferate rapidly. The immune response exhibited breadth because the primed CD4(+) T cells responded to at least three epitopes in the H-2(b) background. Targeting MSLN protein to DEC-205 receptor also resulted in cross-presentation to CD8(+) T cells. Antibody responses against human MSLN were also detected in serum from primed mice by ELISA assays. In summary, targeting of MSLN to DEC-205 improves the induction of CD4(+) and CD8(+) T-cell immunity accompanied by an antibody response. DEC-205-targeting could be valuable for enhancing immunity to MSLN in cancers where this nonmutated protein is expressed.

Enhancement of potent antibody and T-cell responses by a single-dose, novel nanoemulsion-formulated pandemic influenza vaccine

Vaccine shortages are a major obstacle to influenza pandemic preparedness. Increasing vaccine efficiency provides a potentially effective way to overcome this problem. Specifically, using single-dose immunization to induce protective immunity is an attractive approach to emergency/massive vaccination. In this report, we propose a novel nanoemulsion comprised of the bioresorbable polymer, Span ®85, and squalene forming a ready-to-use adjuvant, called PELC. After formulation with PELC, inactivated H5N1 virus was intramuscularly administered to mice via a single injection. The data demonstrate that inactivated virus containing 0.5 μg hemagglutinin (HA) and formulated with PELC induced more potent antigen-specific antibodies, hemagglutination inhibition, and virus neutralization than non-adjuvanted inactivated virus containing 5 μg HA. In addition, T-cell proliferative responses, as well as interferon-γ (IFN-γ) and interleukin-4 (IL-4) secretion were significantly enhanced after immunization with PELC-adjuvanted inactivated virus. These results indicate that PELC can be used for effective single-dose immunization and could thus play an important role in influenza pandemic preparedness.

Feasibility of use of single dose immunization regime in Antibody Induction Method for testing potency of tetanus component in DTP group of vaccines and comparison of validated ELISA with TNT for antibody titration

Conventionally, for estimation of potency of tetanus toxoid by Antibody Induction Method (AIM), two doses (1/10 th of single human dose/dose) of tetanus toxoid given on 0 th and 28 th day, injected subcutaneously into guinea pigs has been recommended. But this assay has a serious drawback of ‘booster effects’ as the animal is immunized on two occasions besides usage of large number of animals. Hence, feasibility of using single dilution given at single occasion was assessed. For this a three dilution regime was used wherein three dilutions of the standard and test tetanus toxoid (TT) were injected subcutaneously into guinea pigs. Postimmunization sera were raised at 4 th and 6 th week and titrated by in-vivo Toxin Neutralization Test (TNT) done in mice and in-vitro indirect enzyme-linked immunosorbent assay (ELISA). For TNT, L+/10 doses of toxin combined with different concentrations of serum samples were inoculated into mice for anti-tetanus antitoxin detection. It was found that if the animal was bled at 4 th week post-immunization, anti-tetanus antibody titers as shown by ELISA were 4-5 times higher than the titers estimated by TNT while at 6 th week ELISA and TN titers were comparable (r 2 =0.999). When the time of bleeding, post-immunization, was increased from 4 th to 6 th week, a 5 fold increase in neutralizing antibody titer was seen. Also, standardized ELISA was found to be of high validity and can be used as an alternate for evaluation of anti-tetanus neutralizing antibodies, thus permitting the in-vitro evaluation of the tetanus antitoxin. Moreover, besides saving mice, ELISA saves five days required for TNT and the pressure on the laboratory is significantly

Chitosan as an adjuvant for parenterally administered inactivated influenza vaccines

The addition of 0.5% of a chitosan derivative to inactivated influenza vaccines injected parenterally resulted in a four or six to tenfold increase in antibody titres after a single-dose or two-dose intramuscular immunization of mice, respectively, in comparison with antibody titres after immunization without chitosan. Chitosan-adjuvanted vaccines enhanced antibody titers against drift variants of A- and B-type human influenza viruses four to six times compared with the vaccines without chitosan. Inactivated avian influenza virus A/H5N2 admixed with chitosan, when administered to mice challenged afterwards with the same virus, showed higher immunogenicity and protective efficacy compared with the antigen without chitosan.

The relative contribution of antibody and CD8 + T cells to vaccine immunity against West Nile encephalitis virus

West Nile virus (WNV) is a mosquito borne, neurotropic flavivirus that causes a severe central nervous system (CNS) infection in humans and animals. Although commercial vaccines are available for horses, none is currently approved for human use. In this study, we evaluated the efficacy and mechanism of immune protection of two candidate WNV vaccines in mice. A formalin-inactivated WNV vaccine induced higher levels of specific and neutralizing antibodies compared to a DNA plasmid vaccine that produces virus-like particles. Accordingly, partial and almost complete protection against a highly stringent lethal intracranial WNV challenge were observed in mice 60 days after single dose immunization with the DNA plasmid and inactivated virus vaccines, respectively. In mice immunized with a single dose of DNA plasmid or inactivated vaccine, antigen-specific CD8(+) T cells were induced and contributed to protective immunity as acquired or genetic deficiencies of CD8(+) T cells lowered the survival rates. In contrast, in boosted animals, WNV-specific antibody titers were higher, survival rates after challenge were greater, and an absence of CD8(+) T cells did not appreciably affect mortality. Overall, our experiments suggest that in mice, both inactivated WNV and DNA plasmid vaccines are protective after two doses, and the specific contribution of antibody and CD8(+) T cells to vaccine immunity against WNV is modulated by the prime-boost strategy.

Rift valley fever virus lacking the NSs and NSm genes is highly attenuated, confers protective immunity from virulent virus challenge, and allows for differential identification of infected and vaccinated animals.

Rift Valley fever (RVF) virus is a mosquito-borne human and veterinary pathogen associated with large outbreaks of severe disease throughout Africa and more recently the Arabian peninsula. Infection of livestock can result in sweeping "abortion storms" and high mortality among young animals. Human infection results in self-limiting febrile disease that in approximately 1 to 2% of patients progresses to more serious complications including hepatitis, encephalitis, and retinitis or a hemorrhagic syndrome with high fatality. The virus S segment-encoded NSs and the M segment-encoded NSm proteins are important virulence factors. The development of safe, effective vaccines and tools to screen and evaluate antiviral compounds is critical for future control strategies. Here, we report the successful reverse genetics generation of multiple recombinant enhanced green fluorescent protein-tagged RVF viruses containing either the full-length, complete virus genome or precise deletions of the NSs gene alone or the NSs/NSm genes in combination, thus creating attenuating deletions on multiple virus genome segments. These viruses were highly attenuated, with no detectable viremia or clinical illness observed with high challenge dosages (1.0 x 10(4) PFU) in the rat lethal disease model. A single-dose immunization regimen induced robust anti-RVF virus immunoglobulin G antibodies (titer, approximately 1:6,400) by day 26 postvaccination. All vaccinated animals that were subsequently challenged with a high dose of virulent RVF virus survived infection and could be serologically differentiated from naïve, experimentally infected animals by the lack of NSs antibodies. These rationally designed marker RVF vaccine viruses will be useful tools for in vitro screening of therapeutic compounds and will provide a basis for further development of RVF virus marker vaccines for use in endemic regions or following the natural or intentional introduction of the virus into previously unaffected areas.