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PubMed2006

Immune responses generated by Lactobacillus as a carrier in DNA immunization against foot-and-mouth disease virus.

To exploit Lactobacillus acidophilus as a carrier in DNA immunization against foot-and-mouth disease virus (FMDV), a recombinant eukaryotic expression plasmid (pRc/CMV2-VP1-Rep. 8014) harboring pRc/CMV2 vector, the FMDV VP1 gene, and a replication origin from Lactobacillus plantarum ATCC 8014 strain was constructed. To detect the VP1 protein, pRc/CMV2-VP1-Rep. 8014 was expressed in PK 15 cells and transfected into a L. acidophilus SW1 strain (L. acidophilus SFMD-1). To evaluate the immunization effect of L. acidophilus SFMD-1, anti-FMDV VP1 antibody, T-cell proliferation, antigen-specific delayed-type hypersensitivity (DTH), and tissue distribution were investigated using intramuscular, intraperitoneal, intranasal, and oral administration delivery routes. The results showed that L. acidophilus SFMD-1 was able to elicit a detectable antibody level on day 21. The VP1 antibody levels induced by L. acidophilus SFMD-1 and commercial inactivated FMDV vaccine rose rapidly to 0.84 and 0.70, respectively, by day 42, then sustained a high level by day 49. The route of administration had an impact on the magnitude of the systemic antigen-specific IgG responses, with intramuscularly applied L. acidophilus SFMD-1 generating the greatest FMDV VP1 antibody response, followed by intraperitoneal, intranasal, and oral administration delivery routes. Using the T-cell proliferation assay, the stimulation index of a group immunized with L. acidophilus SFMD-1 reached 2.78 versus 5.08 in a group immunized with pRc/CMV2-VP1-Rep. 8014 plasmid. Mice immunized with L. acidophilus SFMD-1 were able to induce T-cell-mediated antigen-specific DTH. In addition, the VP1 gene was detected in the muscle, kidney, spleen, and heart, but not in the liver. The results demonstrate clearly that Lactobacillus as a carrier is a promising approach of DNA vaccination, and is a potentially guard against FMDV.

Animals↗

PubMed2004

Mapping of B-cell epitopes in the N-terminal repeated peptides of Anaplasma marginale major surface protein 1a and characterization of the humoral immune response of cattle immunized with recombinant and whole organism antigens.

Major surface protein (MSP) 1a of the genus type species Anaplasma marginale (Rickettsiales: Anaplasmataceae) together with MSP1b forms the MSP1 complex. MSP1a has been shown to be involved in adhesion, infection and tick transmission of A. marginale, as well as to contribute to protective immunity in cattle. A differential antibody response to MSP1a and MSP1b was observed in cattle immunized with A. marginale derived from bovine erythrocytes (anti-MSP1a response) or cultured tick cells (anti-MSP1b response). In this study, we further characterized the MSP1a antibody response of cattle using several immunogens, including recombinant MSP1a (rMSP1a) protein, erythrocyte- or tick cell culture-derived A. marginale, or a combination of tick cell culture-derived A. marginale and rMSP1a. The MSP1a antibody response to all these immunogens was directed primarily against the N-terminal region of MSP1a that contains tandemly repeated peptides, whereas low antibody levels were detected against the C-terminal portion. Linear B-cell epitopes of MSP1a were mapped using synthetic peptides representing the entire sequence of the protein that were prepared by SPOT synthesis technology. Only two peptides in the N-terminal repeats were recognized by sera from immunized cattle. These peptides shared the sequence SSAGGQQQESS, which is likely to contain the linear B-cell epitope that was recognized by the pools of bovine sera. The average differential of antibody titers against MSP1a minus those against MSP1b correlated with lower percent reductions in PCV. A preferential antibody response to MSP1a was observed in cattle immunized with erythrocyte-derived, cell culture-derived plus rMSP1a or rMSP1a alone, and the percent reduction PCV was significantly lower in these cattle as compared with the other immunization groups. These results provide insight into the bovine antibody response against A. marginale and the role of MSP1a in protection of cattle against A. marginale infection.

Anaplasma marginale↗

PubMed2005

The recombinant rabbit hemorrhagic disease virus VP60 protein obtained from Pichia pastoris induces a strong humoral and cell-mediated immune response following intranasal immunization in mice.

Rabbit hemorrhagic disease (RHD) is a contagious and highly lethal viral disease of rabbits that spreads rapidly and infects animals by nasal, conjunctival and oral routes. Therefore, this experiment was undertaken to study the immune response generated after intranasal (i.n.) vaccination with the recombinant VP60 capsid protein from rabbit hemorrhagic disease virus (RHDV) expressed at high levels in Pichia pastoris. Groups of BALB/c mice were immunized with three doses of purified VP60 protein (Group 1), VP60 formulated within the cell debris fraction of the transformed yeast (Group 2) and placebo (Group 3) by intranasal route. Mice were also intramuscularly injected with purified VP60 protein (Group 4). A rapid antibody response specific against rabbit hemorrhagic disease virus was observed in all the experimental groups, except in Group 3, as detected by ELISA. The highest titers were found 60 days after the first immunization. Mice from Group 1 showed the highest IgG response (p<0.05) and the most balanced profile of IgG1, IgG2a and IgG2b subclasses. IgA titers specific to the virus were found only in animals from this group, which also developed the highest specific lymphocyte proliferative response. Interferon-gamma (IFN-gamma) and interleukin-12 (IL-12) gene expression was also detected after an ex vivo-specific stimulation of mice from Groups 1 and 4. These data demonstrated the capacity of VP60 protein expressed in P. pastoris to elicit a potent humoral and cell-mediated immune response following an intranasal immunization scheme.

Administration, Intranasal↗

PubMed2003

Evidence for immune-mediated reduction of viral replication in Macaca nemestrina mucosally immunized with inactivated SHIV(89.6).

Although most HIV-1 infections worldwide result from heterosexual transmission, most vaccine candidates have focused on induction of systemic immunity and protection. We hypothesized that combining systemic priming with mucosal boosting would induce mucosal immunity that would protect from intravaginal challenge. Macaques were primed systemically with recombinant vaccinia viruses and boosted mucosally using inactivated SHIV(89.6) plus adjuvant. Other animals received protein boosts with adjuvant alone. Priming and boosting induced antiviral IgG and IgA antibodies. Such antibodies were induced to a lesser degree in animals receiving boosts alone. Anti-SHIV T cell responses were induced only in the prime-boost animals. Immunized animals and controls were challenged intravaginally with SHIV(89.6) and significant reductions in proviral and viral RNA loads were observed in the prime-boost animals. The boost-only animals did not have significant viral load reductions. These data suggest that cellular immunity was required for protection from intravaginal challenge. This immunization regimen provides a promising lead for vaccine development.

AIDS Vaccines↗

PubMed1998

Oral administration of one dose of cholera toxin induces a systemic immune response prior to a mucosal immune response by a direct presentation in the spleen.

In the present report the results indicate that the oral administration of one dose of CT in rats results in an antibody immune response in the spleen 48 h later, whereas no antitoxin antibody forming cells were found in the Peyer patches (PP), mesenteric lymph node (MLN) and lamina propria (LP) of the small intestine. At this time the main isotype of the antitoxin antibodies in the spleen were IgG and IgM, 5 days after the priming, few antitoxin AFC were observed in the MLN, IgG being the main isotype, whereas no IgM antitoxin AFC were found. At 1 week after priming the number of antitoxin AFC in the MLN reached similar values to those observed in the spleen. When cells from the spleen of rats primed orally with one dose of CT were cultured during 4 days in the presence of inhibitory doses of anti-Ia MAb (OX6), the number of antitoxin AFC was diminished when compared with that observed when cells were cultured in the absence of anti-Ia. The main isotype of antitoxin AFC observed when cells were analyzed after culture was IgM and it was the isotype most affected by the treatment with MAb anti-Ia. These results strongly suggest that an in situ presentation of the antigen did occur in the spleen. On the other hand, when the secondary immune response was studied 48 h after boosting, antitoxin AFC were found in the PP, MLN, SP and LP and 5 days after the booster a 20-30-fold increase was observed in all lymphoid tissues studied, indicating that the secondary immune response found in the spleen was mainly due to the recruitment of memory cells from Peyer's patches. However, when spleen cells were cultured 48 h after the immunization in the presence of inhibitory doses of anti-Ia a little decrease in the number of AFC was observed when compared with the controls (in absence of anti-Ia). The analysis of the antitoxin antibodies in sera and intestinal fluids were in line with the results presented above. The results shown in this report indicate that the systemic immune response observed after the oral administration of CT could be due in part to an in situ presentation of the antigen in the systemic compartments, especially in the spleen.

Administration, Oral↗

PubMed1999

In vivo acquired mechanisms of tumor cells local defense against the host innate immunity effectors: implication in specific antitumor immunity.

As shown earlier, the cells transformed in vitro by several different oncogenes, or spontaneously, during in vivo growth in normal hosts would be gradually replaced by the highly-tumorigenic descendants co-expressing high H2O2-catabolizing and PGE2-releasing activities. Acquisition of (H2O2(CA) + PGE(S)) phenotype provides the cells with local defense mechanisms against the host innate immunity effectors. However, it remained unknown, whether the expression of (H2O2(CA) + PGE(S)) phenotype is implicated in susceptibility of tumor cells expressing tumor-specific transplantation antigens to rejection in immune animals. Here, with the use of SV40 in vitro transformed parental cells, negative in expression (H2O2(CA) + PGE(S)) phenotype, and their in vivo selected descendant tumor cell lines expressing this phenotype, we show that: (1) the rates of in vivo selection of the parental SV40 tumor cells expressing (H2O2(CA) + PGE(S)) phenotype are the same in normal and SV40-immune animals; (2) in vivo selected SV40 tumor cells expressing (H2O2(CA) + PGE(S)) phenotype, although they retain specific immunosensitivity, are 100 times less effectively rejected in SV40-immunized animals, as compared with their in vitro SV40-transformed parental cells. Thus, in vivo acquired immunologically non-specific local mechanisms of tumor cells defense against the host innate immunity effectors, significantly decreases the effectiveness of their specific immunorejection.

Animals↗

PubMed2000

Global immune disregulation in multiple sclerosis: from the adaptive response to the innate immunity.

Increasing evidences show a global immune disregulation in multiple sclerosis (MS). The possible involvement of myelin and non-myelin (auto-)antigens in the autoaggressive process as well as the disregulation of both adaptive and innate immunity challenge the concept of specific immunotherapy. T cells at the boundary between innate and adaptive immunity, whose immunoregulatory role is becoming increasingly clear, have recently been shown to bear relevance for MS pathogenesis. Global immune interventions (and type I interferons may be considered as such) aimed at interfering with both innate and acquired immune responses seem to be a most promising therapeutic option in MS.

Humans↗

PubMed1998

Enhanced immune response after subcutaneous and oral immunization with biodegradable PLGA microspheres.

PLGA microspheres containing bovine serum albumin (BSA) as a model antigen, were prepared by a double emulsion/solvent extraction method and their in vitro characterization was performed. The same microspheres were used in a series of in vivo studies to evaluate the immune response induced after subcutaneous or oral inoculation following different immunization protocols. The in vivo data confirm that the immunogenicity of the albumin is not affected by the encapsulation procedure. The subcutaneous administration of microspheres showed an immune response (serum IgG levels by ELISA) statistically above BSA solution, even when the dose administered was 10 times lower. The adjuvanticity of the microspheres was found to be comparable to that of Freund's complete adjuvant (FCA), but in contrast to FCA they are biocompatible and did not induce any adverse reaction at the site of injection. A single oral administration of the microspheres was not a successful strategy for the induction of a reproducible response. Therefore, microspheres of 1 and 5 micrometer were orally administered on 3 consecutive days and the response obtained showed that the use of a boosting dose was not necessary for the 1 micrometer particles. These results suggest the possibility of simplifying the immunization schedule to a primary immunization if 1 micrometer particles are administered.

Adjuvants, Immunologic↗

PubMed2000

Local and systemic immune responses to combined vibrio cholerae CVD103-HgR and salmonella typhi ty21a live oral vaccines after primary immunization and reimmunization.

The local and systemic antibody responses elicited following concomitant primary immunization and reimmunization with the live oral attenuated Vibrio cholerae CVD103-HgR and Salmonella typhi Ty21a vaccine strains were determined in healthy adult volunteers. A more pronounced serum vibriocidal antibody response was generated after primary immunization compared to reimmunization 2.5 or 3.5 yr later. The seroconversion rate (> or =4-fold rise over baseline) was 81% subsequent to primary immunization versus 57% (p=0.018) and 65% (p=0.639) upon reimmunization at 2.5 and 3.5 yr, respectively. A similar trend was observed for serum anti-S. typhi lipopolysaccharide (LPS) antibodies. After primary immunization, 48% of subjects manifested a significant rise in coproantibody levels to V. cholerae LPS while 60% did so for cholera toxin (CT). Upon reimmunization, the response rate for LPS ranged from 38% at 2.5 yr to 56% at 3.5 yr (p>0.05), while that for CT varied from 31% (p=0. 007) to 50% (p=0.541) at 2.5 and 3.5 yr, respectively. The anti-S. typhi IgA coproantibody response rate was 70% subsequent to primary immunization versus 47% at 2.5 yr (p=0.021) and 63% at 3.5 yr (p=0. 77).

Adult↗

PubMed2001

Enhancement of mucosal immune response against HIV-1 Gag by DNA immunization.

In order to examine the feasibility of Gag-expression DNA as a potential candidate for HIV vaccine using a mouse model, we injected DNA into mice either intramuscularly or by using a gene gun. Both methods induced a low level of antibody production. However, after booster immunization with p24 protein emulsified with complete Freund's adjuvant via a footpad, we found that only the preceding intramuscular DNA immunization induced an anti-Gag Th1-type (IgG(2a)) antibody response, in addition to the enhancement of a Th2-type (IgG(1)) antibody response. Importantly, when mice were boosted intranasally with p24 and cholera toxin, intramuscular DNA injection was found to enhance both systemic and mucosal Gag-specific immune responses. These results indicate that intramuscular DNA immunization confers the inducibility of memory cells, which circulate around various mucosal tissues. Therefore, intramuscular DNA priming, followed by a mucosal booster immunization, could be considered as a regimen applicable to HIV vaccine.

3T3 Cells↗

PubMed2001

Immunization with plasmid DNA encoding the integral membrane protein, Sm23, elicits a protective immune response against schistosome infection in mice.

Schistosomes are helminth parasites infecting at least 200 million people worldwide. In this study, we evaluated the feasibility of using a nucleic acid vaccine to induce protective immune responses to the Schistosoma mansoni integral membrane protein Sm23. C57BL/6 mice were immunized by intramuscular injection in three separate vaccination trials. ELISA and Western Blot analyses indicated that mice immunized with a DNA plasmid construct encoding Sm23 (Sm23-pcDNA) generated specific IgG for Sm23, while sera from mice immunized with the control pcDNA plasmid did not. The vaccine elicited IgG(2a), and IgG(1) antibody isotypes. We also tested the adjuvant activity of IL-12 and IL-4 on humoral responses to Sm23. Co-immunization with plasmid encoding IL-12 did not affect the level of anti-Sm23 IgG(2a), but did reduce the IgG(1) level. In contrast, co-injection with a plasmid encoding IL-4 significantly reduced the level of anti-Sm23 IgG(2a), while the level of IgG(1) was largely unchanged. Importantly, the Sm23-pcDNA vaccine provided statistically significant levels of protection against challenge infection (21-44%, P<0.001-0.02). Co-administration of plasmids encoding either IL-12 or IL-4 did not significantly enhance this protective effect.

Animals↗

PubMed2001

Immunization with a portion of rickettsial outer membrane protein A stimulates protective immunity against spotted fever rickettsiosis.

Two approaches for presentation of a part of the rickettsial outer membrane protein A (OmpA) of Rickettsia rickettsii, namely (1) recombinant Mycobacterium vaccae (rMV) or (2) recombinant DNA vaccine, stimulated protective immunity against a lethal challenge with the closely related bacterium, R. conorii, in mice. After primary immunization with rMV and booster immunization with homologous recombinant protein, 67 and 55% of mice were protected against challenge in two experiments. DNA vaccination with booster recombinant protein immunization protected six out of eight animals from a lethal challenge. Production of IFN-gamma by antigen-exposed T-lymphocytes of DNA vaccine recipients indicated that cellular immunity had been stimulated.

Animals↗

PubMed2003

Effect of vaccine delivery system on the induction of HPV16L1-specific humoral and cell-mediated immune responses in immunized rhesus macaques.

There have been numerous studies to assess the immunogenicity of candidate therapeutic and prophylactic vaccines for human papillomavirus (HPV), but few of them have directly compared different vaccines in an immunologically relevant animal system. In the present study, several vaccine delivery systems (VLPs, chimeric VLPs, plasmid DNA, and a replication incompetent adenoviral vector) expressing HPV16L1 were evaluated for their ability to induce HPV16L1 VLP-specific humoral immune responses, including neutralizing antibodies, and cell-mediated immune responses in rhesus macaques. Monkeys immunized with HPV16L1 VLPs mounted a potent humoral response with strongly neutralizing antibodies and a strong L1-specific Th2 response as measured by IL-4 production by CD4+ T cells. Monkeys immunized with plasmid DNA or an adenoviral vector expressing HPV16L1 showed strong Th1/Tc1 responses as measured by IFN-gamma production by CD4+ and/or CD8+ T cells and potent humoral responses, but only weakly neutralizing antibodies. These data demonstrate that the nature of the immune response against HPV16L1 is dramatically different when it is introduced via different delivery systems. Additionally, these findings support the notion that an HPV16L1 VLP-based vaccine will induce the strongly neutralizing antibodies necessary for effective prophylaxis.

Adenoviridae↗

PubMed2003

Enhanced cellular immune responses to SIV Gag by immunization with influenza and vaccinia virus recombinants.

An effective vaccination strategy against human immunodeficiency virus type 1 (HIV-1) should include the induction of potent cellular immune responses against conserved HIV-1 antigens. We have generated five replication competent recombinant influenza viruses (rFlu/SIV Gag nos. 1-5) expressing different portions of Gag of simian immunodeficiency virus (SIV). Single intranasal immunizations in mice with each rFlu/SIV Gag viruses resulted in different degrees of protection against a challenge with recombinant vaccinia virus expressing SIV Gag. Immunized BALB/c mice had detectable CD8+ T cell responses specific for Gag peptide 185-199 when mice were vaccinated with rFlu/SIV Gag no. 3 virus, and for Gag peptides 281-295 and 285-299 when vaccinated with rFlu/SIV Gag no. 4 virus. Cellular immune responses against SIV Gag were further enhanced by a booster with a recombinant vaccinia virus expressing SIV Gag in both the spleen and local lymph node tissues, resulting in the induction of robust Gag-specific CD8+ T cell responses at both systemic and mucosal levels. We suggest that a prime-boost immunization regimen using recombinant influenza and vaccinia viruses expressing HIV Gag might represent an effective means to induce potent HIV-specific, protective CD8+ T cell responses.

Amino Acid Sequence↗

PubMed1997

Enhancement of immunodeficiency virus-specific immune responses in DNA-immunized rhesus macaques.

In contrast to results obtained with plasmid DNA vectors encoding antigens from viruses such as influenza and hepatitis B, plasmids coding for antigens from primate immunodeficiency viruses have elicited relatively weak antibody responses following gene gun-mediated DNA immunization of rhesus monkeys. In an effort to augment these responses, the importance of the immunization schedule was investigated, as well as the possible synergy that might result from boosting gene gun-primed animals with other routes of immunization. Here we demonstrate that endpoint gp120-specific antibody titers can be enhanced as much as tenfold by reducing the number of immunizations and lengthening the resting period between immunizations. In addition, boosting gene gun-primed animals with either recombinant subunits or gp120-expressing vaccinia recombinants resulted in synergistic responses.

AIDS Vaccines↗

PubMed2000

Effects of IL-12 on immune responses induced by transcutaneous immunization with antigens formulated in a novel lipid-based biphasic delivery system.

The development of non-invasive methods for the delivery of proteins through the permeability barriers, such as the intact skin, will greatly facilitate the administration of human and veterinary vaccines. In the present study we used recombinant Pasteurella haemolytica leukotoxin (Lkt) and hen egg lysozyme (HEL) as model antigens to investigate the ability of transdermal administration of vaccine antigens to induce humoral and cellular responses in mice and to assess the immunomodulatory effects of IL-12 on these antigen-specific immune responses. Mice were immunized by the transdermal route with Lkt or HEL formulated in a novel lipid-based biphasic delivery system (BPDS). Transdermal delivery of Lkt or HEL induced strong polarized Th2 responses characterized by enhancement of antigen-specific IgG1 antibody subclass and predominant induction of antigen specific IL-4 over IFN-gamma in spleen and draining lymph nodes cells. Animals immunized by topical application of formulations containing antigen and IL-12 developed significantly lower antibody titres without significant changes in IL-4 or IFN-gamma secreting cells (SC) in the draining lymph nodes or spleen cells. Our results indicated that application of antigens formulated in BPDS induced antigen-specific immune responses. Furthermore, incorporation of IL-12 to the vaccine formulation influences the induction of antibody responses induced by transdermal immunization. We demonstrated the feasibility of using this technology for the development of non-invasive methods of vaccine administration.

Administration, Cutaneous↗

PubMed1998

Selective compartmental dominance: an explanation for a noninfectious, multifactorial etiology for acquired immune deficiency syndrome (AIDS), and a rationale for ozone therapy and other immune modulating therapies.

The most widely accepted etiological explanation for acquired immune deficiency syndrome (AIDS) currently invokes an infectious model involving the human immunodeficiency virus (HIV). Because this infectious model has failed to meet any conventional criteria for establishing microbial causation, this theory still relies on the high, though not perfect, statistical correlation linking presence of HIV antibodies with patients diagnosed with, and at risk for the syndrome. Many scientists and clinicians now doubt the HIV theory, though, and propose instead a multifactorial causation similar to that seen in cancer and heart disease. In order to discard the HIV model, however, it is necessary to explain the high statistical correlation mentioned above. Recent studies involving cellular mediated immunity and cytokine modulation may explain this statistical relationship without the need to invoke infectious causation, by suggesting certain functional characteristics and feedback loops in the immune system which the author calls selective compartmental dominance (SCD). SCD provides a model in which chronic dominance of the humoral immune compartment secondary to chronic high-dose antigenic challenge results in chronic suppression of the cellular immune compartment. This model predicts that even HIV-negative members of the risk groups are susceptible to AIDS, assigns no special causal role for HIV in AIDS, and suggests a rational course of nontoxic therapy that can potentially reverse cases in the earlier stages.

Acquired Immunodeficiency Syndrome↗

PubMed2003

Analysis of Ig/Ig two-component-determined circulating immune complexes (TCIC) provide new insights into host immunity.

There have been many reports on circulating immune complexes (CIC), but information regarding Ig/Ig two-component-determined circulating immune complexes (TCIC) is not available. In the present study, we measured the levels of heterogeneous Ig/Ig two-component-determined circulating immune complexes (Ig/Ig-TCIC) in healthy individuals and patients with hepatitis A, Graves' disease and gastric cancer. The heterogeneous Ig/Ig-TCIC was dissected into IgM/IgG-TCIC, IgG/IgA-TCIC and IgA/IgM-TCIC by use of coating and detecting antibodies. The current study has been carried out in 383 cases of healthy individuals, 97 cases of hepatitis A, 108 cases of Graves' disease and 126 cases of gastric cancer. We found that changes on the levels of IgM/IgG-TCIC and IgG/IgA-TCIC is a common feature in patients studied in this study. A total of 71.4% and 80% of patients with gastric cancer had decreased IgM/IgG-TCIC and IgG/IgA-TCIC, respectively. In addition, elevated frequencies were found in patients with hepatitis A and Graves' diseases for IgM/IgG-TCIC (51.5% and 38.0%, respectively) and IgG/IgA-TCIC (29.9% and 19.4%, respectively). In summary, our results suggest that Ig/Ig-TCIC may be a hallmark for evaluation of host immunity. Decreased frequency in gastric cancer and increased frequencies in hepatitis A and Graves' disease forms a peculiar trait for detection of IgM/IgG-TCIC and IgG/IgA-TCIC. Our findings have, thus, provided new insights into circulating immune complexes.

Adolescent↗