Pattern of functional antibody activity against Haemophilus influenzae type b (Hib) in infants immunized with diphtheria-tetanus-pertussis/Hib Brazilian combination vaccine

We evaluated the functional activity of Haemophilus influenzae B (Hib) antibodies elicited in a group of infants immunized with the diphtheria-tetanus-pertussis vaccine combined with an Hib vaccine produced totally in Brazil after technological transfer of Hib vaccine production from Glaxo SmithKline, Belgium. Blood samples from immunized infants (N = 985) were collected for the determination of Hib antibodies. Total Ig and IgM and IgG subclasses of antibodies against polyribosyl ribitol phosphate (PRP) were analyzed by ELISA. Almost all vaccinees (97.56 percent, 961/985) developed a strong anti-PRP IgG antibody response (¡Ý1.0 ¦Ìg/mL), while an anti-PRP IgM response was observed in 64.24 percent (634/985) of them (¡Ý0.15 ¦Ìg/mL). Only 18.88 percent (186/985) of the infants in the group with high PRP antibody IgG concentrations (¡Ý1.0 ¦Ìg/mL) developed a high IgM antibody response. Anti-PRP IgG antibody levels were significantly higher than anti-PRP IgM. These results demonstrate the predominance of IgG antibodies over IgM antibodies in response to PRP, with a ratio of 17:1. IgG antibodies were predominantly of the IgG1 subclass. An increase in IgG avidity was also observed during the course of immunization.

Pattern of functional antibody activity against Haemophilus influenzae type B (Hib) in infants immunized with diphtheria-tetanus-pertussis/Hib Brazilian combination vaccine.

We evaluated the functional activity of Haemophilus influenzae B (Hib) antibodies elicited in a group of infants immunized with the diphtheria-tetanus-pertussis vaccine combined with an Hib vaccine produced totally in Brazil after technological transfer of Hib vaccine production from Glaxo SmithKline, Belgium. Blood samples from immunized infants (N = 985) were collected for the determination of Hib antibodies. Total Ig and IgM and IgG subclasses of antibodies against polyribosyl ribitol phosphate (PRP) were analyzed by ELISA. Almost all vaccinees (97.56%, 961/985) developed a strong anti-PRP IgG antibody response (>or=1.0 microg/mL), while an anti-PRP IgM response was observed in 64.24% (634/985) of them (>or=0.15 microg/mL). Only 18.88% (186/985) of the infants in the group with high PRP antibody IgG concentrations (>or=1.0 microg/mL) developed a high IgM antibody response. Anti-PRP IgG antibody levels were significantly higher than anti-PRP IgM. These results demonstrate the predominance of IgG antibodies over IgM antibodies in response to PRP, with a ratio of 17:1. IgG antibodies were predominantly of the IgG1 subclass. An increase in IgG avidity was also observed during the course of immunization.

Detection of Th1/Th2 cytokine signatures in yellow fever 17DD first-time vaccinees through ELISpot assay.

Immunity to yellow fever (YF) is conferred by the interplay of humoral and cellular immune response. Despite the extensive literature on the humoral immune response to the YF vaccine virus, little is known about its cellular immune response to vaccination. The analysis of cytokine production by ex-vivo antigen-stimulated T cells has been considered as a valuable tool for understanding cellular immune response. Thus, we have analyzed two T(H)1/T(H)2 signature cytokines (IFN-gamma and IL-4) from 12 healthy first-time adults vaccinated with YF17DD virus. The cells, harvested on day 0 (before vaccination) and 7, 15 and 30 days after immunization were antigen-stimulated and analyzed by ELISpot. A significant increase in the number of spot-forming cells during the response to YF 17DD live virus stimulation by ELISpot assay was observed. IFN-gamma-and IL-4-producing cells were significantly increased on the 15th day after vaccination in all volunteers. These results presented herein are important for understanding the role of cytokines in the immune response to YF 17DD virus.

Induction of neutralizing antibodies against diphtheria toxin by priming with recombinant Mycobacterium bovis BCG expressing CRM(197), a mutant diphtheria toxin.

BCG, the attenuated strain of Mycobacterium bovis, has been widely used as a vaccine against tuberculosis and is thus an important candidate as a live carrier for multiple antigens. With the aim of developing a recombinant BCG (rBCG) vaccine against diphtheria, pertussis, and tetanus (DPT), we analyzed the potential of CRM(197), a mutated nontoxic derivative of diphtheria toxin, as the recombinant antigen for a BCG-based vaccine against diphtheria. Expression of CRM(197) in rBCG was achieved using Escherichia coli-mycobacterium shuttle vectors under the control of pBlaF*, an upregulated beta-lactamase promoter from Mycobacterium fortuitum. Immunization of mice with rBCG-CRM(197) elicited an anti-diphtheria toxoid antibody response, but the sera of immunized mice were not able to neutralize diphtheria toxin (DTx) activity. On the other hand, a subimmunizing dose of the conventional diphtheria-tetanus vaccine, administered in order to mimic an infection, showed that rBCG-CRM(197) was able to prime the induction of a humoral response within shorter periods. Interestingly, the antibodies produced showed neutralizing activity only when the vaccines had been given as a mixture in combination with rBCG expressing tetanus toxin fragment C (FC), suggesting an adjuvant effect of rBCG-FC on the immune response induced by rBCG-CRM(197). Isotype analysis of the anti-diphtheria toxoid antibodies induced by the combined vaccines, but not rBCG-CRM(197) alone, showed an immunoglobulin G1-dominant profile, as did the conventional vaccine. Our results show that rBCG expressing CRM(197) can elicit a neutralizing humoral response and encourage further studies on the development of a DPT vaccine with rBCG.

Immunostimulatory effects of polar glycopeptidolipids of Mycobacterium chelonae for inactivated rabies vaccine.

Humoral and cellular immune responses were analyzed with Fuenzalida-Palacios rabies vaccine associated with pGPL-Mc, polar glycopeptidolipids extracted from Mycobacterium chelonae, aiming at its use as adjuvant. These results were compared to those obtained with BCG, a well-known immunostimulator, under the same conditions. Rabies vaccine plus pGPL-Mc (2.5 mg/kg) induced a significant increase in serum neutralizing activity, in vitro lymphocyte proliferation (spontaneous, specific and mitogen stimulation) and delayed type hypersensibility. In addition, pGPL-Mc, as well as BCG, enhanced the vaccine potency. Our results support further studies to encourage the use of pGPL-Mc as an immunostimulator of veterinary vaccines, before consideration for human vaccines.

[Immunogenicity of the avirulent strain RV194-2 of the rabies virus in mice]. / Imunogenicidade da cepa avirulenta RV194-2 do vírus rábico em camundongos.

RV194-2 rabies virus, an avirulent mutant of CVS strain, induces an inapparent infection limited to the central nervous system (CNS) in adult mice inoculated intracerebrally. This fact suggest that immune response of the host is able to eliminate the virus in CNS. For this reason, we have studied the induction of interferon and the humoral immune responses in BALB/c mice after RV194-2 inoculation. These mice presented high levels of interferon in the plasma and in the brain, with elevated levels of neutralizing antirabies antibodies. The 2-5A synthetase, an enzyme marker of interferon action, was analyzed in the brain of inoculated animals. Its enhancement in parallel to the interferon production in the brain, showed biochemical evidence that this interferon is active. Forty five days after RV194-2 virus inoculation, mice were protected against a challenge with the CVS virulent strain. The results presented herein show that RV194-2 strain has a high level of immunogenicity.

Lymphocyte subsets and cell proliferation analysis in rabies-infected mice.

Alterations to lymphocyte subsets in the thymus and spleen of rabies-infected mice were investigated in a kinetic study covering the entire course of infection. Changes in the levels of thymic Thy1.2 cells were found to be proportional to total thymocyte depletion, while thymic CD4-/CD8- and B cell subsets were observed to increase in comparison to the total number of cells. The same was found to be true of CD4 and CD8 thymocytes, but only after the first clinical signs of disease had appeared. Meanwhile, drastic reductions in the number of CD4+/CD8+ thymocytes occurred during the course of infection. In the spleen, on the other hand, alterations to splenocyte subsets were not selective. Thymocytes expressing high levels of CD4 or CD8 markers were detected shortly before the death of the animals. Following the appearance of rabies symptomatology, lymphocyte proliferation decreased in comparison to the control. Thus, our results demonstrate that the thymus is the most injured lymphoid organ in cases of murine rabies infection.

Interferon production and immune response induction in apathogenic rabies virus-infected mice.

Pathogenic parental rabies virus strain CVS (challenge virus standard) and its apathogenic variant RV194-2 were shown to differ in their ability to induce interferon (IFN) and immune response of the host. After intracerebral inoculation, IFN and antibody production was higher in the RV194-2 virus-infected mice than in the CVS infection. The enhancement of 2-5A synthetase activity, an IFN-mediated enzyme marker, showed biochemical evidence that IFN is active in both apathogenic and pathogenic infections. On the other hand, spontaneous proliferation in vitro of thymocytes and splenocytes from CVS virus-infected mice was strongly inhibited in contrast to the RV194-2 infection. In the CVS infection, the thymocyte proliferation was more affected than the splenocyte proliferation. However, in the RV194-2 infection, the thymocyte proliferation was higher than of the splenocytes. These results suggest a better performance of T-cell response to the RV194-2 infection than to the CVS infection. This fact can be critical for an enhancement of antibody production in the apathogenic infection and subsequent virus clearance from the brain of RV194-2 virus-infected mice.

Relationship between apoptosis and thymocyte depletion in rabies-infected mice.

The apoptosis of thymocytes from rabies-infected mice was investigated in a kinetic study covering the entire course of the infection. For this study, BALB/c mice (6-7-week old females) were inoculated intracerebrally with 100 LD50 of Challenge Virus Strain, a fixed rabies virus strain, and three animals were sacrificed per time point to remove thymuses. When thymocytes were fixed, stained with propidium iodide and analyzed by flow cytometry, a distinct subpopulation of cells was observed below the G0/G1 region, denoted as the A0 region. Cells in this region presented reduced fluorescence, and nuclear DNA fragmentation. The accumulation of cells in the A0 region, after infection, progressively increased, reaching 12% for unfractionated thymocytes, 62% for thymocytes from the 60% Percoll interface and 32% for thymocytes recovered at the 100% Percoll interface. This finding, observed only in thymocytes from infected mice, demonstrates a clear modification of chromatin condensation in these cells, suggesting the occurrence of an apoptotic process during rabies infection.

Relationship between apoptosis and thymocyte depletion in rabies-infected mice

The apoptosis of thymocytes from rabies-infected mice was investigated in a kinetic study covering the entire course of the infection. For this study, BALB/c mice (6-7-week old females) were inoculated intracerebrally with 100 LD50 of Challenge Virus Strain, a fixed rabies virus strain, and three animals were sacrificed per time point to remove thymuses. When thymocytes were fixed, stained with propidium iodide and analyzed by flow cytometry, a distinct subpopulation of cells was observed below the G0/G1 region, denoted as the A0 region. Cells in this region presented reduced fluorescence, and nuclear DNA fragmentation. The accumulation of cells in the A0 region, after infection, progressively increased, reaching 12 for unfractionated thymocytes, 62 for thymocytes from the 60 Percoll interface and 32 for thymocytes recovered at the 100 Percoll interface. This finding, observed only in thymocytes from infected mice, demonstrates a clear modification of chromatin condensation in these cells, suggesting the occurrence of an apoptotic process during rabies infection.

2-5A synthetase expression in mice infected with Trypanosoma cruzi.

This study describes the production and action of interferon in mice infected with Colombian and Y strain T. cruzi. The production of interferon was monitored by an in vitro assay of plasma and extract of spleen, lung and heart for interferon activity. The action of interferon in mice was assessed by measuring an interferon-mediated enzyme activity, 2-5A synthetase. Infected mice (strain Balb/c) were sacrificed at different time intervals, and the level of this enzyme was measured in extracts of spleen, lung and heart. Colombian strain infection induced higher levels of interferon than Y strain under the same conditions; consequently, a greater increase in 2-5A synthetase induction was observed in the former of the two strains. These results suggest that interferon produced by T. cruzi infected mice is active, since a variety of organs respond to its presence by producing elevated levels of 2-5A synthetase.

20 alpha-hydroxysteroid dehydrogenase expression in a murine virus-induced myeloproliferative syndrome.

The myeloproliferative sarcoma virus (MPSV) infection in DBA/2 mice leads to important quantitative and qualitative changes in their hemopoiesis. These findings suggest a disturbance in the production and action of a certain hemopoietic factor similar to IL3. Here, we show that the level of the 20 alpha-hydroxysteroid dehydrogenase (20 alpha-SDH) expression, which can be induced by IL3, is dramatically increased in spleen and thymus of MPSV-infected mice. Our results suggest that quantification of 20 alpha-SDH activity can be used to indicate abnormal production of a growth factor similar to IL3 in hemopoietic system diseases.

The effect of interferon treatment in rabies prophylaxis in immunocompetent, immunosuppressed, and immunodeficient mice.

The development of rabies is modulated by many interacting factors, most of which are dependent on the host immune response. For this reason, we studied the action of interferon (IFN) treatment on street rabies virus infection in mice, immunocompetent or immunosuppressed with cyclophosphamide. In immunocompetent mice, paralysis of hind limbs is the first symptom characteristic of rabies disease before weight loss and general prostration leading to death. Paralysis does not occur in immunosuppressed mice, which develop a shaggy hair and eventually lose weight and die. Administration of interferon (10(5) units, intraperitoneally) 1 h after virus inoculation and every 24 h led to a delay in the onset of first disease signs, but in general did not rescue immunocompetent or immunosuppressed mice from death. In both types of mice, rabies virus production in the brain was reduced by 1 log in response to IFN treatment. In immunocompetent mice treated with IFN, there was a significant increase of antibody synthesis against rabies virus. As expected, antibody synthesis in immunosuppressed mice was almost negligible. However, in mice treated with IFN and cyclophosphamide there was still significant antibody synthesis specific for rabies virus. IFN administered intravenously, subcutaneously, or intraperitoneally crosses the blood-brain barrier to cause enhanced levels of the two double-stranded RNA-dependent enzymes, the protein kinase and 2',5'-oligoadenylate (2-5A) synthetase in the brain. However in spite of this effect, IFN treatment seems to be unable to prevent the evolution of rabies disease in immunocompetent and immunosuppressed mice. Since the suppressing effect of cyclophosphamide is nonselective on both the cellular and humoral immune responses of mice, we investigated the action of IFN in rabies virus-infected athymic nude mice, which lack T cells. Athymic nude mice infected with street rabies virus become cachectic and die without any apparent symptom of paralysis of hind limbs. IFN treatment (administered as above) protected nude mice against rabies infection. Three months after virus inoculation and 2 months after the end of IFN treatment, 7 of 8 IFN-treated mice remained in perfect health. These results illustrate that the efficacy of IFN treatment against the evolution of rabies disease in mice is dependent on the suppression of the T-cell-mediated immune response of the host.

Neutralization of interferon produced early during rabies virus infection in mice.

Inoculation of mice (strain C3H/He) with a purified preparation of fixed rabies virus led to the production of interferon with two different peaks of activity detectable in the plasma: an early peak 24 h after inoculation followed by another peak on the 7th day after infection. The level of splenic 2-5A synthetase was enhanced in parallel with the pattern of interferon activity. Neutralization of the first peak of interferon activity by anti-mouse alpha/beta interferon globulin blocked the induction of splenic 2-5A synthetase and modified the development of disease. Infected mice given anti-interferon globulin had a significantly shorter morbidity period than normally infected mice. These results suggest that interferon produced early after virus inoculation plays a role in the onset of rabies disease.

Comparison of the effects of rabies virus infection and of combined interferon and poly(I).poly(C) treatment on the levels of 2',5'-adenyladenosine oligonucleotides in different organs of mice.

Intracellular levels of 2',5'-adenyladenosine oligonucleotides were analyzed in different organs of mice during the course of a rabies virus infection. Phosphorylated and nonphosphorylated 2',5'-adenyladenosine oligonucleotides were measured by radioimmunoassay and analyzed further by HPLC. As the infection progressed, concentrations of phosphorylated 2',5'-adenyladenosine oligonucleotides increased strongly, reaching their maxima late in the infection. In contrast, concentrations of the nonphosphorylated 2',5'-adenyladenosine oligonucleotides decreased. A similar phenomenon was observed in spleens analyzed at intervals after treatment of noninfected mice with interferon and poly(I).poly(C) and to a lesser extent after treatment of noninfected mice with interferon and poly(I).poly(C) and to a lesser extent after treatment with poly(I).poly(C) alone, but not after treatment with interferon alone. The products which accumulated during virus infection were primarily phosphorylated dimers whereas during combined interferon and poly(I).poly(C) treatment, the entire range of phosphorylated molecules from dimer to pentamer was present. These data show that infection of mice with rabies virus provokes both the induction and the activation of 2-5A synthetase, as does interferon and poly(I).poly(C) treatment. However, our data indicate that the intracellular products are different in the two situations: the species active on the nuclease were only detected in interferon- and poly(I).poly(C)-treated mice. The absence of molecules able to activate the 2-5A-dependent nuclease in virus-infected mice might well be one of the reasons why the interferon system is ineffective in rabies virus infection.