Enhanced protection against respiratory influenza A infection in mice by liposome-encapsulated antibody.

Liposome-mediated passive immunity was evaluated for its efficacy in the prophylaxis and treatment of influenza A/PR/8 virus infection in mice. A mouse LD50 protection model was developed using a polyclonal anti-influenza A antibody which demonstrated strong reactivity against the mouse-adapted virus in a fluorogenic enzyme immunoassay and in an in vitro plaque assay. Using liposomes as an antibody carrier system, the delivery of antibody to the lungs was optimized. For mice given the antiviral antibody intranasally 24 h prior to challenge with 10 LD50 of mouse-adapted influenza A/PR/8 virus, the survival rate at 14 days post-challenge was 60%. However, when mice were given antibody encapsulated within liposomes, the survival rate increased to 100%. In the treatment of mice preinfected with 10 LD50 of the virus, mice were fully protected (100% survival rate) when treated within 8 hr post-infection with free unencapsulated antibody, or within 12 hr with liposome-encapsulated antibody. It is postulated that the improved therapeutic and prophylactic efficacies of the antiviral antibody may be attributed to enhanced delivery as well as retention of antibody molecules in the lungs when liposomes are used as antibody carrier system.     

Inclusion of CpG adjuvant with plasmid DNA coding for NcGRA7 improves protection against congenital neosporosis

The present study showed that incorporation of CpG adjuvant into plasmid DNA coding for NcGRA7 antigen resulted in a twofold increase in the level of protection against congenital transfer of Neospora caninum. The level of protection was considerably higher than that observed in pups born from dams immunized with nonrecombinant plasmid.     

Enhanced protection of mice against Japanese encephalitis virus infection by combinations of monoclonal antibodies to glycoprotein E

In the present study, the protective effect of various combinations of four monoclonal antibodies (MAbs) to glycoprotein E (gpE) of Japanese encephalitis virus (JEV) on the JEV-infected mice was studied. The MAbs were characterized as hemagglutination-inhibition-positive and JEV-specific (Hs). In the protective experiment, mice were first administered single MAbs or their combinations intraperitoneally (i.p.) and 24 hrs later infected with the virus intracerebrally (i.c.). The results showed that single MAbs protected the mice to the extent of 45-65%, while combinations of two or three MAbs gave 85-90% or 100% protection, respectively. The enhanced effect of combinations of several Hs MAbs might be due to the sharing of neutralization epitopes recognized by the Hs MAbs. These results suggested that a combination of at least three epitopes represented by the Hs MAbs should be included in an effective JEV vaccine.     

DNA vaccine using Mycobacterium bovis Ag85B antigen induces partial protection against experimental infection in BALB/c mice

Bovine tuberculosis is a major cause of economic loss in countries where it is endemic, and in some countries, it may be a significant zoonotic disease problem. Therefore, new strategies for vaccine development are required, and among them, genetic immunization has potential value. The main goal of this study was to test the Mycobacterium bovis Ag85B gene as a DNA vaccine following challenge with an M. bovis virulent strain (ATCC 19274). Groups of BALB/c mice (n = 10) were immunized four times intramuscularly with the pCI-Ag85B construct or the pCI vector alone as the control. High titers of total immunoglobulin G (IgG), IgG1, and IgG2a anti-Ag85B were measured in pCI-Ag85B immunized mice when compared to the pCI control group. Regarding cellular immunity, significant levels of gamma interferon (IFN-gamma) (1,100 +/- 157 pg/ml) and tumor necrosis factor alpha (650 +/- 42 pg/ml) but not interleukin-4 were detected in splenocyte culture supernatants of pCI-Ag85B-vaccinated mice following stimulation with recombinant Ag85B. Further, the main source of IFN-gamma is CD8(+) T cells, as demonstrated by intracellular cytokine staining. As far as protection, a significant reduction in bacterial load in spleens (P < 0.05) was detected in pCI-Ag85B-immunized mice compared to the pCI vector control group. The results obtained here suggest that use of the Ag85B DNA vaccine is a promising strategy to control M. bovis infection due to its ability to induce a Th1 type of immune response. However, protective efficacy needs to be improved, since partial protection was achieved in spleens but not in lungs of vaccinated mice.     

SAG1 is a host-targeted antigen for protection against toxoplasma gondii infection.

We previously reported that SAG1 transgenic (tg) mice have an elevated susceptibility resulting from their inability to elicit strong Th1-based protection against Toxoplasma gondii infection. Here, we demonstrate that SAG1 tg mice were protected against T. gondii infection, characterized by a decline in IFN-gamma levels, following administration of a lethal dose of T. gondii. Moreover, immunization with T. gondii homogenate conferred protection and induced production of IgG, with IgG1 and IgG2a subclasses driven by Th2 and Th1 responses, respectively, in both SAG1 tg and wild-type (wt) mice. IgG titers were significantly higher from day 10 after immunization in wt mice compared to those in SAG1 tg mice. There were no significant differences observed in levels of IgG1 in both groups. However, significantly lower IgG2a titers were measured in the sera from SAG1 tg mice on days 10, 15 and 20. IFN-gamma levels in sera were significantly lower in SAG1 tg mice compared to those in wt mice on day 20 after immunization. When challenged with a lethal dose of the Beverley strain of T. gondii, 80 and 100% survival rates were observed in SAG1 tg and wt mice, respectively, indicating that SAG1 tg mice were protected to a lesser extent from challenge due to the decrease in protective immunity. These results suggest that SAG1 plays a critical role in eliciting protection, hence a target antigen for the development of protective Th1-based responses against T. gondii infection in mice.     

Active protection against rotavirus infection of mice following intraperitoneal immunization.

Active immunity to rotavirus has been demonstrated following oral inoculation with live virus but little is known about the effects of parenteral immunization. In this study, adult mice were immunized by intraperitoneal (ip) inoculation with live rotaviruses and later orally challenged with murine rotavirus (EDIM) to measure active immunity against infection. Three doses of EDIM (8 micrograms/dose) given intraperitoneally (ip) provided full protection against EDIM infection, whether administered with or without Freund's adjuvant. Only partial protection was found when the quantity of immunogen was reduced to < 2 micrograms/dose. Reduction of the number of doses from three to one (8 micrograms/dose), however, still resulted in protection of all mice. Significant protection was also observed after inoculation with one or three doses (2 micrograms/dose) of heterologous rotaviruses. Protection provided by the heterologous strains did not correlate with neutralizing antibody to EDIM, which indicated that neutralizing antibody to the challenge virus was not required for protection. uv-Inactivated EDIM also provided significant protection against EDIM, thus demonstrating that viral replication was not required for protection. These results suggest that parenteral immunization may be an effective method to vaccinate against rotavirus disease.     

Comparison of antigen expression from plasmid DNA in tumor-free and antigen-expressing tumor-bearing mice

Reduced antigen expression after DNA vaccination could be a factor that limits its therapeutic effects in antigen-expressing tumor-bearing hosts. To examine whether the presence of an antigen-positive tumor results in the destroy of antigen-expressing cells, pCMV-OVA plasmid expressing ovalbumin (OVA), a model antigen, was intradermaly injected to tumor-free mice and those bearing an EG7-OVA tumor, which stably expresses OVA. For a quantitative evaluation of the elimination of antigen-expressing cells, a plasmid independently expressing firefly luciferase and OVA (pCMV-Luc/OVA) was injected to mice that had been untreated or administered with pCMV-OVA, and luciferase activity was measured one week after the administration. In both tumor-free and tumor-bearing mice, a pre-injection of pCMV-OVA reduced the luciferase expression from pCMV-OVA/Luc compared with a pre-injection of an empty plasmid, suggesting that an immune response to antigen-expressing cells is induced. In tumor-bearing mice, OVA-specific humoral and cellular immune responses were detected even before the vaccination, but such responses had no significant effects on the luciferase expression from pCMV-OVA/Luc. These results indicate that DNA vaccination can induce antigen-specific immune response even in tumor-bearing mice, although it is not strong enough to reject the tumor burden.     

Partial protection against Brucella infection in mice by immunization with nonpathogenic alphaproteobacteria

Previous findings indicate that Brucella antigens and those from nonpathogenic alphaproteobacteria (NPAP) are cross-recognized by the immune system. We hypothesized that immunization with NPAP would protect mice from Brucella infection. Mice were immunized subcutaneously with heat-killed Ochrobactrum anthropi, Sinorhizobium meliloti, Mesorhizobium loti, Agrobacterium tumefaciens, or Brucella melitensis H38 (standard positive control) before intravenous challenge with Brucella abortus 2308. Cross-reacting serum antibodies against Brucella antigens were detected at the moment of challenge in all NPAP-immunized mice. Thirty days after B. abortus challenge, splenic CFU counts were significantly lower in mice immunized with O. anthropi, M. loti, and B. melitensis H38 than in the phosphate-buffered saline controls (protection levels were 0.80, 0.66, and 1.99 log units, respectively). In mice immunized intraperitoneally with cytosoluble extracts from NPAP or Brucella abortus, protection levels were 1.58 for the latter, 0.63 for O. anthropi, and 0.40 for M. loti. To test whether the use of live NPAP would increase protection further, mice were both immunized and challenged by the oral route. Immunization with NPAP induced a significant increase in serum immunoglobulin G (IgG), but not serum or fecal IgA, against Brucella antigens. After challenge, anti-Brucella IgA increased significantly in the sera and feces of mice orally immunized with O. anthropi. For all NPAP, protection levels were higher than those obtained with systemic immunizations but were lower than those obtained by oral immunization with heat-killed B. abortus. These results show that immunization with NPAP, especially O. anthropi, confers partial protection against Brucella challenge. However, such protection is lower than that conferred by immunization with whole Brucella or its cytosoluble fraction.     

Vaccination with plasmid DNA encoding TSA/LmSTI1 leishmanial fusion proteins confers protection against Leishmania major infection in susceptible BALB/c mice

We have recently shown that a cocktail containing two leishmanial recombinant antigens (LmSTI1 and TSA) and interleukin-12 (IL-12) as an adjuvant induces solid protection in both a murine and a nonhuman primate model of cutaneous leishmaniasis. However, because IL-12 is difficult to prepare, is expensive, and does not have the stability required for a vaccine product, we have investigated the possibility of using DNA as an alternative means of inducing protective immunity. Here, we present evidence that the antigens TSA and LmSTI1 delivered in a plasmid DNA format either as single genes or in a tandem digene construct induce equally solid protection against Leishmania major infection in susceptible BALB/c mice. Immunization of mice with either TSA DNA or LmSTI1 DNA induced specific CD4(+)-T-cell responses of the Th1 phenotype without a requirement for specific adjuvant. CD8 responses, as measured by cytotoxic-T-lymphocyte activity, were generated after immunization with TSA DNA but not LmSTI1 DNA. Interestingly, vaccination of mice with TSA DNA consistently induced protection to a much greater extent than LmSTI1 DNA, thus supporting the notion that CD8 responses might be an important accessory arm of the immune response for acquired resistance against leishmaniasis. Moreover, the protection induced by DNA immunization was specific for infection with Leishmania, i.e., the immunization had no effect on the course of infection of the mice challenged with an unrelated intracellular pathogen such as Mycobacterium tuberculosis. Conversely, immunization of BALB/c mice with a plasmid DNA that is protective against challenge with M. tuberculosis had no effect on the course of infection of these mice with L. major. Together, these results indicate that the protection observed with the leishmanial DNA is mediated by acquired specific immune response rather than by the activation of nonspecific innate immune mechanisms. In addition, a plasmid DNA containing a fusion construct of the two genes was also tested. Similarly to the plasmids encoding individual proteins, the fusion construct induced both specific immune responses to the individual antigens and protection against challenge with L. major. These results confirm previous observations about the possibility of DNA immunization against leishmaniasis and lend support to the idea of using a single polygenic plasmid DNA construct to achieve polyspecific immune responses to several distinct parasite antigens.     

Antigen-induced protection against infection withToxocara vitulorum larvae in mice

Larvae ofToxocara vitulorum hatched and migrated in the tissues of normal mice. Larvae survived in reasonable numbers, particularly in the liver and, to a lesser extent, in the lungs and kidneys, for at least 4–7 days and in muscles, albeit only in low numbers, for at least 3 weeks. Oral infection of mice on three or more occasions withT. vitulorum eggs induced protection against a challenge infection with eggs ofT. vitulorum. Prior parenteral immunization of mice with a variety ofT. vitulorum soluble antigens (extracts, excretions/secretions, or perienteric fluid and their fractions) from adult parasites and/or infective larvae induced statistically significant protection against infection. The most effective protective immunogens were three or more injections with perienteric fluid from adults (100% protection) and excretions/secretions from infective larvae ofT. vitulorum (>92% protection).     

Complete protection against lethal Toxoplasma gondii infection in mice immunized with a plasmid encoding the SAG1 gene

Infection with the protozoan parasite Toxoplasma gondii is transmitted to humans from infected animals by tissue cysts and oocysts excreted by cats. Immunization with inactivated parasites or recombinant proteins has at best shown partial protection. We constructed a plasmid expressing the SAG1 surface antigen of T. gondii, p1tPASAG1, and showed that animals immunized with the plasmid produce anti-SAG1 antibodies which recognize the native SAG1. Mice immunized with p1tPASAG1 showed 80 to 100% protection against challenge with the non-cyst-producing, virulent RH isolate, compared to an 80% mortality in mice immunized with empty plasmid, which is the greatest efficacy of any vaccine against T. gondii produced so far. The SAG1 molecule was analyzed for potential cytotoxic T-lymphocyte (CTL) epitopes, and four peptides with the best fit were synthesized. The ability of the peptides to stimulate gamma interferon production by CD8(+) T cells from p1tPASAG1-immunized mice was tested in an ELISPOT assay, and one new CTL epitope was identified. Adoptive transfer of CD8(+) T cells from p1tPASAG1-immunized to na?ve mice showed partial protection. In conclusion, DNA vaccination with p1tPASAG1 gave effective protection in mice against T. gondii infection and the protection could be adoptively transferred by purified CD8(+) T cells.     

Protection against Gram-negative infection by 'super-active' antigen.

'Super-active' antigens modified antigens released from bacteria which had been phagocytosed and killed by human leucocytes, were found to induce protective responses in mice within 24 h of immunization. At the earliest time (24 h) when immunized mice were protected against lethal intrapertoneal (i.p.) challenge by the bacteria from which which the 'super-active' antigens were made (Proteus mirabilis) the leucocytes of peripheral blood from immunized mice showed enhanced phagocytosis and killing of autologous bacteria and there was an increase in the number of lymphocytes producing anti-proteus antibody. Another mouse protective factor inducing transient protection lasting 1-2 days against lethal i.p. challenge by P. mirabilis was found in preparations of lysed heman leucocytes not engaged in phagocytosis. Burned mice, immunized with 'super-active' antigen preparations were protected against lethal invasive proteus infection, inoculated on to the burn surface, 2 h after burning and immunization.     

A DNA vaccine coding for the Brucella outer membrane protein 31 confers protection against B. melitensis and B. ovis infection by eliciting a specific cytotoxic response

The development of an effective subunit vaccine against brucellosis is a research area of intense interest. The outer membrane proteins (Omps) of Brucella spp. have been extensively characterized as potential immunogenic and protective antigens. This study was conducted to evaluate the immunogenicity and protective efficacy of the B. melitensis Omp31 gene cloned in the pCI plasmid (pCIOmp31). Immunization of BALB/c mice with pCIOmp31 conferred protection against B. ovis and B. melitensis infection. Mice vaccinated with pCIOmp31 developed a very weak humoral response, and in vitro stimulation of their splenocytes with recombinant Omp31 did not induced the secretion of gamma interferon. Splenocytes from Omp31-vaccinated animals induced a specific cytotoxic-T-lymphocyte activity, which leads to the in vitro lysis of Brucella-infected macrophages. pCIOmp31 immunization elicited mainly CD8(+) T cells, which mediate cytotoxicity via perforins, but also CD4(+) T cells, which mediate lysis via the Fas-FasL pathway. In vivo depletion of T-cell subsets showed that the pCIOmp31-induced protection against Brucella infection is mediated predominantly by CD8(+) T cells, although CD4(+)T cells also contribute. Our results demonstrate that the Omp31 DNA vaccine induces cytotoxic responses that have the potential to contribute to protection against Brucella infection. The protective response could be related to the induction of CD8(+) T cells that eliminate Brucella-infected cells via the perforin pathway.     

Enhanced protection by use of a combination of anticapsule and antilipopolysaccharide monoclonal antibodies against lethal Escherichia coli O18K5 infection of mice.

To study antibody-mediated protection against Escherichia coli peritonitis in BALB/c mice, monoclonal antibodies (MAbs) were generated against the capsule (K5) and the lipopolysaccharide (O18) of E. coli. Flow cytometric analysis with two selected immunoglobulin M MAbs revealed that bacteria were antigenically heterogeneous. Arbitrarily, three subpopulations in E. coli O18K5 cultures could be distinguished by double immunofluorescence. A subpopulation bound only the anti-K5 MAb, and another subpopulation bound only the anti-O18 MAb. An intermediate subpopulation, however, bound both MAbs. In agreement with this result, combinations of both MAbs enhanced phagocytosis of fluorescein isothiocyanate-labeled bacteria by human polymorphonuclear leukocytes and mouse macrophage J774 cells as well. In protection experiments, combinations of both MAbs, preincubated with 3 50% lethal doses of E. coli O18K5, protected all mice upon intraperitoneal challenge. Relatively high doses of either MAb alone proved to be not fully protective in this infection model. Protection of mice by the combination of MAbs was associated with significantly lower (P < 0.02) tumor necrosis factor levels in serum 90 min after challenge compared with any other treatment group. Similarly, prophylactic administration of MAbs yielded significantly lower (P < 0.01) tumor necrosis factor levels in mice that received the combination of MAbs than in any other treatment group.     

IFN-alpha1 plasmid construct affords protection against HSV-1 infection in transfected L929 fibroblasts.

The purpose of the present study was to evaluate the resistance against herpes simplex virus type 1 (HSV-1) using an interferon-alpha1 (IFN-alpha1) transgene in specifically targeted cells in vitro. Transfection of mouse fibroblast L929 cells with an IFN-alpha1 plasmid construct reduced viral load and viral gene expression in a time-dependent fashion. Supernatants from IFN-alpha1-transfected cells augmented natural killer (NK) cell activity, and such an effect was antagonized with neutralizing antibody to IFN-alpha/beta. In addition, transfected cells displayed an increase in the IFN inducible genes (2',5'-oligoadenylate synthetase [2',5'-OAS], T cell-specific guanine nucleotide triphosphate-binding protein, IFN regulatory factor 1 [IRF-1], and major histocompatibility complex [MHC] class I) compared with plasmid vector-treated controls. Collectively, these results show that IFN-alpha1 transfection of cells in vitro induces or upregulates a spectrum of IFN-regulated genes involved in the direct or indirect antiviral action of this cytokine. In addition, the transgene significantly increases the resistance of transfected cells in vitro to HSV-1 infection.     

Enhanced resistance against encephalomyocarditis virus infection in mice, induced by a nonviable Mycobacterium tuberculosis oil-droplet vaccine.

Female C57B1/10 mice injected intravenously (i.v.) with nonviable Mycobacterium tuberculosis Jamaica cells associated with oil-droplet emulsions (WCV) were highly resistant to the i.v. injection of encephalomyocarditis virus (EMCV). Resistance to infection (87% survival) was detected from 1 week to at least 12 weeks after injection of WCV. Mice vaccinated i.v. also were resistant to intraperitoneal, subcutaneous, or intramuscular virus challenge, but were not resistant to intracranial challenge. Mice vaccinated intraperitoneally also were resistant to virus infection, whereas WCV administered intramuscularly or subcutaneously did not protect mice from virus injected by any route. Less than 50% of WCV mice that survived virus challenge possessed serum anti-EMCV-neutralizing antibody (less than 1:10), and none had detectable (less than 1:10) serum interferon. Interferon was not detected in sera of WCV mice from 4 to 144 h after i.v. injection of EMCV. Studies concerning the effects of WCV on EMCV infection suggest that mice may be protected by mechanisms that inhibit early viral replication and spread of virus to the central nervous system.     

Animal model of rotavirus infection in rabbits—protection obtained without shedding of viral antigen

Summary A small animal model was developed in order to investigate the pathogenesis and immunology of rotavirus infections and to study the interaction of different virus strains. Seronegative rabbits of the breed French Lop were used. Two rabbit rotavirus strains, belonging to the same serotype, were used: 82/311F and R-2, both isolated during diarrhoeal outbreaks in commercial rabbitries. The animals were inoculated orally. The viral shedding and the serological response was monitored by ELISA. Initially six weeks old kits were given four different doses of strain R-2. With doses ranging from 1 × 10³ to 1 × 10⁶ TCID₅₀ all animals seroconverted, but for the lowest dose no viral excretion could be detected. No clinical symptoms were observed. Subsequently the age periods during which the animals were susceptible to the strain R-2 was investigated. The rabbits seroconverted and shed rotavirus antigen, independent of age of six or 22 weeks. None of the animals had diarrhoea. Administration of strain 82/311 F did not result in viral shedding, independently of dose, but all the animals seroconverted. It was also shown for the strain R-2 that when challenging with the same strain four weeks post inoculation that the animals were protected; no viral shedding was detected at the second infection. Strain 82/311 F gave protection against R-2 when the rabbits were challenged four weeks post inoculation.     

DNA-mediated immunization of mice with plasmid encoding HBs antigen.

In order to develop an experimental DNA vaccine for the prevention and treatment of hepatitis B virus infection, hepatitis B virus surface antigen (HBsAg) DNA was subcloned into an E. coli-eukaryotic cell shuttle vector and was expressed in the Baculovirus expression system. Intramuscular, intradermal, and intraperitoneal injections of 30 microg of the plasmid DNA expressing HBsAg induced humoral and cellular immune responses in ICR mice. The first IgG antibodies were detected after ten days and specific IgG antibody titers peaked after two months of a single intramuscular DNA injection. Anti-HBs antibody titers gradually increased and peaked at four months following intradermal DNA injection, and in case of intraperitoneal injection they peaked at seven months. Generation of HBs-specific helper T lymphocytes was also investigated through the production of interleukin-2 by T helper cells. Boosting effects of HBs DNA were investigated without much results. In general, DNA-mediated HBs immunization induced humoral and cellular immune responses in mice that appears to simulate immune responses in human during the course of HBV vaccination.