Efficacy of vaccination strategies against intranasal challenge with Brucella abortus in BALB/c mice

Brucellosis is a zoonotic disease affecting 500,000 people worldwide annually. Inhalation of aerosol containing a pathogen is one of the major routes of disease transmission in humans. Currently there are no licensed human vaccines available. Brucella abortus strain RB51 is a USDA approved live attenuated vaccine against cattle brucellosis. In a mouse model, strain RB51 over-expressing superoxide dismutase (SOD) administered intraperitoneally (IP) has been shown to be more protective than strain RB51 against an IP challenge with B. abortus pathogenic strain 2308. However, there is lack of information on the ability of these vaccine strains to protect against intranasal challenge. With the long-term goal of developing a protective vaccine for animals and people against respiratory challenge of Brucella spp., we tested a number of different vaccination strategies against intranasal infection with strain 2308. We employed strains RB51 and RB51SOD to assess the efficacy of route, dose, and prime-boost strategies against strain 2308 challenge. Despite using multiple protocols to enhance mucosal and systemic protection, neither rough RB51 vaccine strains provided respiratory protection against intranasal pathogenic Brucella infection. However, intranasal (IN) administration of B. abortus vaccine strain 19 induced significant (p ≤ 0.05) pulmonary clearance of strain 2308 upon IN challenge infection compared to saline. Further studies are necessary to address host-pathogen interaction in the lung microenvironment and elucidate immune mechanisms to enhance protection against aerosol infection.     

Smallpox DNA vaccine delivered by novel skin electroporation device protects mice against intranasal poxvirus challenge

Previously, we demonstrated that an experimental smallpox DNA vaccine comprised of four vaccinia virus genes (4pox) administered by gene gun elicited protective immunity in mice challenged with vaccinia virus, and in nonhuman primates challenged with monkeypox virus (Hooper JW, et al. Smallpox DNA vaccine protects nonhuman primates against lethal monkeypox. J Virol 2004;78:4433-43). Here, we report that this 4pox DNA vaccine can be efficiently delivered by a novel method involving skin electroporation using plasmid DNA-coated microneedle arrays. Mice vaccinated with the 4pox DNA vaccine mounted robust antibody responses against the four immunogens-of-interest, including neutralizing antibody titers that were greater than those elicited by the traditional live virus vaccine administered by scarification. Moreover, vaccinated mice were completely protected against a lethal (>10LD(50)) intranasal challenge with vaccinia virus strain IHD-J. To our knowledge, this is the first demonstration of a protective immune response being elicited by microneedle-mediated skin electroporation.     

Recombinant A27 protein synergizes with modified vaccinia Ankara in conferring protection against a lethal vaccinia virus challenge

Highly attenuated modified vaccinia virus Ankara (MVA) is being considered as a safer alternative to conventional smallpox vaccines such as Dryvax or ACAM 2000, but it requires higher doses or more-frequent boosting than replication-competent Dryvax. Previously, we found that passive transfer of A27 antibodies can enhance protection afforded by vaccinia immune globulin (VIG), which is derived from Dryvax immunized subjects. Here we investigated whether protective immunity elicited by MVA could be augmented by prime-boost or combination immunizations with a recombinant A27 (rA27) protein. We found that a prime/boost immunization regimen with rA27 protein and MVA, in either sequence order, conferred protection to mice challenged with a lethal dose of vaccinia virus strain Western Reserve (VV-WR), compared to no protection after immunizations with a similar dose of either MVA or rA27 alone. Moreover, protection was achieved in mice primed simultaneously with combination of both MVA and rA27 in different vaccination routes, without any boost, even though MVA or rA27 alone at the same dose gave no protection. These findings show that rA27 can synergize with MVA to elicit robust protection that has a dose-sparing effect on MVA and can accelerate protection by eliminating the need for a booster dose.     

Adsorption of recombinant poxvirus L1-protein to aluminum hydroxide/CpG vaccine adjuvants enhances immune responses and protection of mice from vaccinia virus challenge

The stockpiling of live vaccinia virus vaccines has enhanced biopreparedness against the intentional or accidental release of smallpox. Ongoing research on future generation smallpox vaccines is providing key insights into protective immune responses as well as important information about subunit-vaccine design strategies. For protein-based recombinant subunit vaccines, the formulation and stability of candidate antigens with different adjuvants are important factors to consider for vaccine design. In this work, a non-tagged secreted L1-protein, a target antigen on mature virus, was expressed using recombinant baculovirus technology and purified. To identify optimal formulation conditions for L1, a series of biophysical studies was performed over a range of pH and temperature conditions. The overall physical stability profile was summarized in an empirical phase diagram. Another critical question to address for development of an adjuvanted vaccine was if immunogenicity and protection could be affected by the interactions and binding of L1 to aluminum salts (Alhydrogel) with and without a second adjuvant, CpG. We thus designed a series of vaccine formulations with different binding interactions between the L1 and the two adjuvants, and then performed a series of vaccination-challenge experiments in mice including measurement of antibody responses and post-challenge weight loss and survival. We found that better humoral responses and protection were conferred with vaccine formulations when the L1-protein was adsorbed to Alhydrogel. These data demonstrate that designing vaccine formulation conditions to maximize antigen–adjuvant interactions is a key factor in smallpox subunit-vaccine immunogenicity and protection.     

A subunit vaccine candidate derived from a classic H5N1 avian influenza virus in China protects fowls and BALB/c mice from lethal challenge

In recent years, numerous human infections with avian influenza viruses in Asia have raised the concern that the next influenza pandemic is imminent. The most effective way to combat human avian influenza is through vaccination of the public. In this study, we developed an influenza A recombinant protein (rH5HA) directed against the hemagglutinin (HA) of a classic H5N1 high pathogenic avian influenza virus isolated in South China in 1996. Following purification of the recombinant protein expressed from a baculovirus expression system, we evaluated the efficiency of rH5HA on specific pathogen free (SPF) chicken, commercial chicken, and in BALB/c mice in an infection-protection model. The results demonstrated that rH5HA induced antibody responses and provided full protection in both SPF chickens and commercial chickens. Protective immunity was generated within 2 weeks in chickens as young as 7-day post-hatch using a minimum amount of rH5HA protein (2 μg/bird/vaccination). The serum antibody generated from rH5HA immunization was protective and lasted more than 6 months. Our data also demonstrated that rH5HA immunization protected BALB/c mice from a lethal challenge with pathogenic avian influenza virus. These results suggested that vaccination with rH5HA could be a vaccine candidate for the control of H5N1 avian influenza in poultry, in mice, and potentially in other mammals including human.     

Intranasal immunisation with recombinant adenovirus vaccines protects against a lethal challenge with pneumonia virus of mice

Pneumonia virus of mice (PVM) infection of BALB/c mice induces bronchiolitis leading to a fatal pneumonia in a dose-dependent manner, closely paralleling the development of severe disease during human respiratory syncytial virus infection in man, and is thus a recognised model in which to study the pathogenesis of pneumoviruses. This model system was used to investigate delivery of the internal structural proteins of PVM as a potential vaccination strategy to protect against pneumovirus disease. Replication-deficient recombinant human adenovirus serotype 5 (rAd5) vectors were constructed that expressed the M or N gene of PVM pathogenic strain J3666. Intranasal delivery of these rAd5 vectors gave protection against a lethal challenge dose of PVM in three different mouse strains, and protection lasted for at least 20 weeks post-immunisation. Whilst the PVM-specific antibody response in such animals was weak and inconsistent, rAd5N primed a strong PVM-specific CD8⁺ T cell response and, to a lesser extent, a CD4⁺ T cell response. These findings suggest that T-cell responses may be more important than serum IgG in the observed protection induced by rAd5N.     

布鲁氏菌104M液体气溶胶免疫BALB/c小鼠的有效性评价和安全性研究

目的 评价布鲁氏菌104M液体气溶胶肺递送途径免疫BALB/c小鼠有效性和安全性。方法 随机选取6～8周龄BALB/c雌鼠,分别经肺递送、滴鼻和皮下注射3种途径免疫接种布鲁氏菌104M,于免疫后第4、8、16、24周观察并记录小鼠的症状、检测小鼠体重、脾重、脾脏载菌量及肺匀浆、血清的抗体和细胞因子。待鼠脾脏载菌完全清除,用布鲁氏菌A19液体气溶胶肺递送途径攻毒。结果 各组实验小鼠均未见异常症状;体重无显著下降;攻毒前,脾重没有明显变化;攻毒后,免疫组小鼠脾重显著低于空白对照组（P<0.05）：液体气溶胶肺递送：实验组（0.26±0.16）g<空白对照组（0.40±0.19）g,滴鼻：实验组（0.21±0.11）g<空白对照组（0.28±0.19）g,皮下注射：实验组（0.14±0.02）g<空白对照组（0.30±0.18）g。随着免疫时间的增长,免疫组小鼠脾脏载菌量呈下降趋势,第20周完全清除。攻毒后2周（免疫24周）,所有小鼠脾脏载菌均显著增加,各免疫组脾载菌量均显著低于空白对照组（P<0.05）：脾载菌量以log₁₀菌落形成单位（colony-forming units,CFU）/g计数并统计分析,液体气溶胶肺递送：实验组（4.49±0.13）<空白对照组（6.90±0.46）;滴鼻：实验组（3.59±1.06）<空白对照组（7.08±0.14）;皮下注射：实验组（3.00±2.03）<空白对照组（6.81±0.34）。布鲁氏菌104M激发了BALB/c小鼠细胞免疫和体液免疫反应。在免疫后第4周,检测到104M特异性抗体IgG、IgM、IgA,第8周达到高峰,攻毒后再次显著上升。各免疫组血清和肺匀浆中IFN-γ和IL-18浓度在攻毒前,均显著高于空白对照组（P<0.05）,攻毒后,各免疫组血清IFN-γ和IL-18浓度均低于空白对照组（P<0.05）,而肺匀浆细胞因子浓度在攻毒前后均持续高于空白对照组（P<0.05）。结论 液体气溶胶肺递送途径是一种有效的免疫途径,表现出有效的保护作用;104M未引起小鼠体重减轻,相对安全,但在小鼠体内存活时间较长,引起小鼠轻度脾脏肿大,有一定的残余毒力。     

Prevention of vertical transmission of Neospora caninum in BALB/c mice by recombinant vaccinia virus carrying NcSRS2 gene

Neospora caninum infection is the major cause of bovine abortion. To develop a vaccine against N. caninum infection, recombinant vaccinia viruses carrying NcSRS2 and NcSAG1 genes (vv/Nc-p43 and vv/Nc-p36, respectively) were constructed and were tested in a mouse model. Vaccination of dams with vv/Nc-p43 appeared to confer effective protection against vertical transmission to offspring, though that with vv/Nc-p36 only provided partial protection. Moreover, the vv/Nc-p43 vaccination provoked cellular immune responses and antibody production against N. caninum. In conclusion, it is expected that vv/Nc-p43 can be used as an effective live vaccine to prevent vertical transmission of N. caninum in natural hosts.     

Recombinant M2e outer membrane vesicle vaccines protect against lethal influenza A challenge in BALB/c mice

Currently approved influenza vaccines predominantly protect through antibodies directed against the highly variable glycoprotein hemagglutinin (HA), necessitating annual redesign and formulation based on epidemiological prediction of predominant circulating strains. More conserved influenza protein sequences, such as the ectodomain of the influenza M2 protein, or M2e, show promise as a component of a universal influenza A vaccine, but require a Th1-biased immune response for activity. Recently, recombinant, bacterially derived outer membrane vesicles (OMVs) demonstrated potential as a platform to promote a Th1-biased immune response to subunit antigens. Here, we engineer three M2e-OMV vaccines and show that all elicit strong IgG titers, with high IgG2a:IgG1 ratios, in BALB/c mice. Additionally, the administration of one M2e-OMV construct containing tandem heterologous M2e peptides (M2e4xHet-OMV) resulted in 100% survival against lethal doses of the mouse-adapted H1N1 influenza strain PR8. Passive transfer of antibodies from M2e4xHet-OMV vaccinated mice to unvaccinated mice also resulted in 100% survival to challenge, indicating that protection is driven largely via antibody-mediated immunity. The potential mechanism through which M2e-OMVs initiated the immune response was explored and it was found that the constructs triggered TLR1/2, TLR4, and TLR5. Our data indicate that OMVs have potential as a platform for influenza A vaccine development due to their unique adjuvant profile and intrinsic pathogen-mimetic nature.     

Maternal immunization with a DNA vaccine candidate elicits specific passive protection against post-natal Zika virus infection in immunocompetent BALB/c mice

Zika virus (ZIKV) infection is closely associated in the fetus with microcephaly and in the adults with Guillain-Barré syndrome and even male infertility. It is an urgent international priority to develop a safe and effective vaccine that offers protection to both women of childbearing age and their children. In this study, female immunocompetent BALB/c mice were immunized with a DNA-based vaccine candidate, pVAX1-ZME, expressing the prM/E protein of ZIKV, and the immunogenicity for maternal mice and the post-natal protection for suckling mice were evaluated. It was found that administration with three doses of 50 μg pVAX1-ZME via in vivo electroporation induced robust ZIKV-specific cellular and long-term humoral immune responses with high and sustained neutralizing activity in adult mice. Moreover, using a maternal immunization protocol, neutralizing antibodies provided specific passive protection against ZIKV infection in neonatal mice and effectively inhibited the growth delay. This vaccine candidate is expected to be further evaluated in higher animals, and maternal vaccination shows great promise for protecting both women of childbearing age and their offspring against post-natal ZIKV infection. The vaccinated mothers and ZIKV-challenged pups provide key insight into Zika vaccine evaluation in an available fully immunocompetent animal model.     

Intranasal immunization with tetanus toxoid and CNF1 as a new mucosal adjuvant protects BALB/c mice against lethal challenge

Although often requiring the development of efficient adjuvants, needle-free mucosal delivery of vaccine is of major interest as a strategy of mass immunization against infectious diseases. We report that mucosal immunization against tetanus toxoid through nasal route, together with active cytotoxic necrotizing factor 1 (CNF1), elicits a specific and long lasting anti-tetanus toxin response, comprising seric IgG and IgA, as well as mucosal IgA. Immunized mice were protected against a challenge with lethal doses of tetanus toxin (10 × LD₅₀). The Rho GTPase activating toxin CNF1 is thus an attractive mucosal adjuvant candidate for nasal vaccines.     

Clonal vaccinia virus grown in cell culture fully protects monkeys from lethal monkeypox challenge

The potential use of smallpox as an agent of bioterrorism has renewed interest in the development of a modern vaccine capable of replacing the standard Dryvax vaccine. Vaccinia virus (ACAM2000), clonally isolated from Dryvax and manufactured in cell culture, was tested for immunogenicity and protective activity in a non-human primate model. Cynomolgus monkeys vaccinated with ACAM2000, Dryvax, or ACAM2000 diluent (control) were challenged 2 months post-vaccination with a lethal, intravenous dose of monkeypox virus. ACAM2000 proved immunogenic and efficacious in protecting against lethal monkeypox challenge, as evident from a lack of post-challenge viral replication, and the absence of any significant clinical signs attributable to monkeypox infection. This protection correlated (with) neutralizing antibody titers equivalent to those generated in the Dryvax group post-vaccination, as well as a similar significant increase in the presence of neutralizing antibodies post-challenge. Control animals showed no signs of vaccine-induced seroconversion, displayed post-challenge tissue-associated viral replication and viremia, and developed severe monkeypox-specific clinical symptoms. The protective efficacy of ACAM2000 was found to be equivalent to the currently approved vaccine, Dryvax.     

Ebola virus glycoprotein Fc fusion protein confers protection against lethal challenge in vaccinated mice

Ebola virus is a Filoviridae that causes hemorrhagic fever in humans and induces high morbidity and mortality rates. Filoviruses are classified as “Category A bioterrorism agents”, and currently there are no licensed therapeutics or vaccines to treat and prevent infection. The Filovirus glycoprotein (GP) is sufficient to protect individuals against infection, and several vaccines based on GP are under development including recombinant adenovirus, parainfluenza virus, Venezuelan equine encephalitis virus, vesicular stomatitis virus (VSV) and virus-like particles. Here we describe the development of a GP Fc fusion protein as a vaccine candidate. We expressed the extracellular domain of the Zaire Ebola virus (ZEBOV) GP fused to the Fc fragment of human IgG1 (ZEBOVGP-Fc) in mammalian cells and showed that GP undergoes the complex furin cleavage and processing observed in the native membrane-bound GP. Mice immunized with ZEBOVGP-Fc developed T-cell immunity against ZEBOV GP and neutralizing antibodies against replication-competent VSV-G deleted recombinant VSV containing ZEBOV GP. The ZEBOVGP-Fc vaccinated mice were protected against challenge with a lethal dose of ZEBOV. These results show that vaccination with the ZEBOVGP-Fc fusion protein alone without the need of a viral vector or assembly into virus-like particles is sufficient to induce protective immunity against ZEBOV in mice. Our data suggested that Filovirus GP Fc fusion proteins could be developed as a simple, safe, efficacious, and cost effective vaccine against Filovirus infection for human use.     

Immunization with pseudorabies virus harboring Fc domain of IgG makes a contribution to protection of mice from lethal challenge

To enhance the efficacy of an inactivated vaccine against pseudorabies virus (PRV), we evaluated the adjuvant properties of Fc domain of IgG. A cell line expressing mouse IgG Fc chimera on its surface was established. We found that when PRV was propagated in the cells expressing the Fc chimera, PRV virion incorporated the Fc. Immunization of BALB/c mice with inactivated PRV harboring Fc, which had been propagated in the cells expressing Fc on its surface, induced higher antibody production against PRV and protected mice more effectively from lethal challenge of virulent strain, comparing to the immunization with normal inactivated virus. Virus harboring Fc has a great potential as a new inactivated vaccine.     

A heterologous prime-boost regime using DNA and recombinant vaccinia virus expressing the Leishmania infantum P36/LACK antigen protects BALB/c mice from cutaneous leishmaniasis.

A heterologous prime-boost vaccination with DNA vectors and vaccinia virus recombinants (VVr) has been shown to enhance specific cellular immune responses and to elicit significant protection against pathogens in animal models. In this study, we have analyzed, in the leishmaniasis cutaneous murine model, the effectiveness of this prime-boost strategy by immunizing with a DNA vector followed by boost with a VVr expressing the same Leishmania infantum P36/LACK antigen. After DNA priming and VVr boost, we challenged susceptible BALB/c mice with live L. major promastigotes, and examined the increase in footpad lesion size and parasite load in draining lymph nodes. Compared to controls, we observed reduction of up to 70% in lesion size and 1000-fold in parasite load. DNA prime-VVr boost before challenge elicited a Th1 type immune response in spleen cells from immunized animals. This DNA/VVr vaccination approach could be of utility in the prophylaxis against leishmaniasis.     

Protection induced in mice against a lethal orthopox virus by the Lister strain of vaccinia virus and modified vaccinia virus Ankara (MVA)

The Lister (Elstree) strain of vaccinia virus, used in Israel for vaccination against smallpox, was studied in tissue cultures and in a mouse model. The virus failed to reach the brain of the mice when inoculated intranasally at a dose of 500,000 plaque forming units, but was lethal for 50% of them, when injected intracranially. Lower doses of virus injected intracranially caused some weight loss initially, but later the mice completely recovered. Modified vaccinia virus Ankara (MVA), when infected intranasally, did not spread beyond the lungs to other organs of the mice. Even when the mice were inoculated with MVA intracranially, they were not affected. Significant protection against a lethal dose of an orthopoxvirus was obtained in mice following immunization with the Lister strain, while larger doses and repeated vaccination procedure, were required with MVA. The Lister virus stock applied in Israel, was found to be heterogeneous in its plaque morphology. Two variants isolated from it, showed significant attenuation for mice, when inoculated intranasally and intracranially, as compared to a third variant and to the unpurified stock of the virus.     

A modified HIV challenge assay in mice by using luciferase-expressing vaccinia virus

In this study, we developed a simple and sensitive assay in mice for a challenge experiment by using a recombinant vaccinia virus dual-expressing antigen (HIV Env gp160) and firefly luciferase. This assay can detect the vaccine effect at real-time in vivo by using a small amount of mouse serum. The luciferase activity in mouse serum was in agreement with the viral titer in the ovary. This assay would be applicable as a challenge model for infectious diseases.     

Vaccination with Brucella recombinant DnaK and SurA proteins induces protection against Brucella abortus infection in BALB/c mice

The immunogenicity and protective efficacy of recombinant SurA (rSurA) and rDnaK from Brucella spp. were evaluated in BALB/c mice. Immunization with rSurA in adjuvant induced a vigorous immunoglobulin G (IgG) response, with higher IgG2a than IgG1 titers. In addition, after in vitro stimulation with rSurA, spleen cells from rSurA-immunized mice produced interleukin-2 (IL-2), interferon (IFN)-gamma, IL-4 and IL-5. Immunization with rDnaK plus adjuvant induced a strong humoral response resulting in similar anti-rDnaK IgG titers than immunization with rDnaK alone. IgG2a titers predominated over IgG1 in mice injected with rDnaK alone or rDnaK plus adjuvant. Spleen cells from mice immunized with rDnaK plus adjuvant secreted IFN-gamma and IL-2 upon stimulation with rDnaK and induced a specific cytotoxic response. On the contrary, mice immunized with rDnaK alone did not exhibit a specific T helper or cytotoxic response in vitro. Mice given rSurA or rDnaK with adjuvant exhibited a significant degree of protection whereas immunization with rDnaK alone induced a low but still statistically significant level of protection against B. abortus infection. All studied vaccines were less protected than mice immunized with H38 or B. abortus strain 19 control vaccines. Altogether these results suggest that rSurA or rDnaK induce partial protection against B. abortus infection and could be useful candidates for the development of subunit vaccines against brucellosis.     

Plasma beads loaded with Candida albicans cytosolic proteins impart protection against the fungal infection in BALB/c mice

The development of a prophylactic vaccine against systemic candidiasis, employing Candida albicans cytosolic proteins (Cp) as antigen and fibrin cross-linked plasma beads as an antigen bearing dual delivery system is described. Groups of mice were administered either with free Cp, or Cp entrapped in plasma beads, Cp entrapped in liposomes or liposome encapsulated Cp further entrapped in plasma beads. Humoral immunity was studied by measuring the anti-Cp antibody titers in the sera of the immunized animals. Induction of cell-mediated immunity was assessed by delayed type hypersensitivity (DTH), NO production, up-regulation of co-stimulatory molecules viz. CD80, CD86 on APCs on one hand and T-cells proliferation as well as induction of IFN-γ and IL-4 on the other. The efficacy of various vaccine formulations in protecting mice against a lethal challenge with C. albicans, was assessed by determining animal survival rate and fungal burden in the systemic circulation and vital organs. Among various Cp-based vaccines investigated, the preparation containing liposomized Cp entrapped in plasma beads imparted superior protection in the immunized mice as compared to other antigens delivery systems.