Vaccine strategies to overcome maternal antibody mediated inhibition of measles vaccine

A vaccine that is effective in the presence of maternally derived virus neutralizing antibodies and can be administered successfully at an early age, would be favoured over the presently used live attenuated measles vaccines. With the advent of new molecular and immunological techniques, several options for the development of new generation vaccines, fulfilling these criteria, have arisen. We have recently evaluated the efficacy of recombinant vaccinia virus- and iscom-based candidate vaccines, presenting the F and H proteins of measles virus, in macaques with passively transferred virus neutralizing macaque antibodies. The data indicate that the further exploration of the potential of iscom based measles vaccines should be encouraged.     

Secondary but not primary T cell responses are enhanced in CTLA-4-deficient CD8+ T cells

Negative as well as positive co-stimulation appears to play an important role in controlling T cell activation. CTLA-4 has been proposed to negatively regulate T cell responses. CTLA-4-deficient mice develop a lymphoproliferative disorder, initiated by the activation and expansion of CD4+ T cells. To assess the function of CTLA-4 on CD8+ T cells, CTLA-4(-/-) animals were crossed to an MHC class I-restricted 2C TCR transgenic mouse line. We demonstrate that although the primary T cell responses were similar, the CTLA-4-deficient 2C TCR+ CD8+ T cells displayed a greater proliferative response upon secondary stimulation than the 2C TCR+ CD8+ T cells from CTLA-4 wild-type mice. These results suggest that CTLA-4 regulates antigen-specific memory CD8+ T cell responses.     

Enhanced B-1 cell development, but impaired IgG antibody responses in mice deficient in secreted IgM

The role of endogenous natural IgM in promoting the adaptive Ab response was investigated in newly constructed mutant mice in which B cells do not secrete IgM but still express surface IgM and IgD and undergo class switching to express other Ig isotypes. While the mutant mice had relatively normal numbers of conventional B (B-2) cells in all tissues examined, unexpectedly, B-1 cells in the peritoneum and spleen were approximately threefold more abundant. The elevated levels of B-1 cells were already detectable at 4 wk of age and were stably maintained throughout life. The levels of serum IgG2a, IgG3, and IgA were also elevated in the mutant mice at an early age. IgG2a response to a T cell-independent Ag was augmented, whereas IgG Ab responses to suboptimal doses of a T cell-dependent Ag were impaired. The latter defect was associated with fewer splenic germinal centers, impaired Ab affinity maturation, and less Ag trapping on follicular dendritic cells. Together, these findings demonstrate a physiologic role of natural IgM in the feedback regulation of B-1 cell development, the regulation of IgG2a production, and the promotion of efficient B-2 cell Ab responses.     

Selective inhibition of T cell proliferation but not expression of effector function by human alveolar macrophages

BACKGROUND: Alveolar macrophages are thought to play an important part in regulating lung immune responses. While it is clear that human alveolar macrophages suppress T cell proliferation in vitro, the mechanisms by which this is achieved are not clear, nor is it known whether alveolar macrophages also inhibit other aspects of T cell function. METHODS: Peripheral blood mononuclear cells were stimulated with phytohaemagglutinin or house dust mite allergen, and cultured with variable numbers of autologous alveolar macrophages obtained by bronchoalveolar lavage from 20 normal subjects. RESULTS: Alveolar macrophages induced a reversible inhibition of T cell proliferation in response to both mitogen and allergen stimulation, with the latter being considerably more susceptible to inhibition. This was achieved via heterogenous mechanisms, involving both soluble factors derived from alveolar macrophages and cell-cell contact. Despite inhibiting proliferation, alveolar macrophages had little or no effect on T cell calcium flux, the characteristic changes in CD3, CD2, CD28 and interleukin-2 (IL-2) receptor expression which accompany normal T cell activation, and IL-2 and interferon gamma secretion. In contrast, alveolar macrophages inhibited the tyrosine phosphorylation of proteins which may be involved in IL-2 receptor-associated signal transduction. CONCLUSIONS: The immunoregulatory properties of alveolar macrophages are relatively selective, allowing T cell activation and cytokine secretion while inhibiting T cell proliferation within the lung.     

Effects of maternal antibodies on protection and development of antibody responses to human rotavirus in gnotobiotic pigs

Although maternal antibodies can protect against infectious disease in infancy, they can also suppress active immune responses. The effects of circulating maternal antibodies, with and without colostrum and milk antibodies, on passive protection and active immunity to human rotavirus (HRV) were examined in gnotobiotic pigs. Pigs received intraperitoneal injections of high-titer serum (immune pigs [groups 1 and 2]) from immunized sows, low-titer serum from naturally infected sows (control pigs [groups 3 and 4]), or no serum (group 5). Immune or control colostrum and milk were added to the diet of groups 2 and 4, respectively. After inoculation (3 to 5 days of age) and challenge (postinoculation day [PID] 21) with virulent HRV, the effects of maternal antibodies on protection (from diarrhea and virus shedding), and on active antibody responses (measured by quantitation of antibody-secreting cells [ASC] in intestinal and systemic lymphoid tissues by ELISPOT) were evaluated. Groups 1 and 2 had significantly less diarrhea and virus shedding after inoculation but higher rates of diarrhea and virus shedding after challenge than did groups 3 and 5. Group 1 and 2 pigs had significantly fewer immunoglobulin A (IgA) ASC in intestinal tissues at PID 21 and at postchallenge day (PCD) 7 compared to group 5. Significantly fewer IgG ASC were present in the intestines of group 2 pigs at PID 21 and PCD 7 compared to group 5. There was a trend towards fewer ASC in intestinal tissues of group 2 than group 1, from PID 21 on, with significantly fewer IgA ASC at PCD 7. IgG ASC in the duodenum and mesenteric lymph nodes of group 3 and 4 pigs were significantly fewer than in group 5 at PCD 7. These decreases in ASC emphasize the role of passive antibodies in impairing induction of ASC rather than in merely suppressing the function of differentiated B cells. To be successful, vaccines intended for populations with high titers of maternal antibodies (infants in developing countries) may require higher titers of virus, multiple doses, or improved delivery systems, such as the use of microencapsulation or immune stimulating complexes, to overcome the suppressive effects of maternal antibodies.     

Preferential but not exclusive T cell receptor V beta chain utilization of myelin basic protein and peptide-specific T cell clones in mice

The repertoire of T cell receptor (TCR) V beta chain utilization was investigated in PL/J, CXJ-1, SJL/J and B10.S-->SJL/J chimeric mice in response to either myelin basic protein (MBP) or the strain-specific encephalitogenic peptide. Our analysis showed that there was an overlapping predominance in the TCR V beta gene utilization in the MBP-specific responses, which were independent of the major histocompatibility complex (MHC) class II haplotype present, and the immunodominant peptide region recognized in these different strains. In those mice having the TCR V beta b haplotype (PL/J, CXJ-1, and the B10.S-->SJL chimera) either the TCR V beta 4, 8, and 13 or the TCR V beta 4, 6, and 13 predominated. In contrast, in mice with TCR V beta a haplotype (SJL/J) V beta 4, 6, and 17a were found. However, the quantitative distribution of these preferentially utilized TCR V beta chains in each strain was defined by the MHC class II haplotype and the immunodominant peptide recognized. The expression of the V beta 8 gene product in the peripheral TCR repertoire did not always correlate with predominant V beta 8 utilization in the MBP-specific response.     

Docosahexaenoic and eicosapentaenoic acids inhibit human lymphoproliferative responses in vitro but not the expression of T cell surface activation markers

The effects of polyunsaturated fatty acids (PUFAs: docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids) on induced lymphocyte proliferation and expression of CD25alpha chain of interleukin-2 receptor, CD71 and HLA-DR were investigated. PUFAs had no effect on phytohaemagglutinin (PHA)-induced lymphocyte agglutination, but they strongly inhibited the lymphoproliferative response to PHA. This inhibitory effect is PUFA dose-dependent and seems to be more potent with DHA than EPA, Pre-incubation experiments showed that lymphocytes cultured with PUFAs for 6 h then washed and exposed to PHA, still inhibited lymphocyte proliferation. The authors also showed that this inhibitory activity was time dependent but became nonsignificant when PUFAs were added after 48 h lymphocyte culture. The addition of excess exogenous human recombinant rIL-2 partly restored PHA-lymphocyte proliferation inhibited by EPA but not by DHA. On the other hand, the authors showed that PUFAS did not inhibit IL-2 stimulated lymphocyte proliferation. The addition of PUFAs to cell culture medium had no inhibitory action on the PHA-induced lymphocyte expression of CD25, CD71 and HLA-DR. Furthermore, this effect appeared independent of eicosanoid synthesis or peroxide formation. Indeed, the inclusion of aspirin and vitamin E in the culture medium did not prevent the inhibitory effects of PUFAs on lymphocyte proliferation. Regardless of the mechanism of action, the inhibitory effect of PUFAs on activated lymphocytes may explain why some clinical trials of fish oil supplemented diets containing high amounts of DHA and EPA have been successful in improving the health status of patients suffering from inflammatory and autoimmune disorders.     

Prevention of diabetes but not insulitis in NOD mice injected with antibody to CD4

Non-obese diabetic (NOD) mice were injected with a rat monoclonal antibody to CD4 from birth every two weeks through 6 months of age. These animals gained weight normally but < 11% of their spleen T cells were CD4+, compared with 28% of CD4+ in controls injected with polyclonal rat IgG. The reduction in CD4 cell percentage was associated with a reduction in the number of cells in the thymus and spleen following the injection. CD4+ cells which survived the injections were nevertheless able to enter cell cycle when stimulated by Con A. None of the CD4-treated NOD mice became diabetic by 6 months of age and none of the animals studied histologically at this time had insulitis. At 9 months of age (three months after stopping the CD4 injections) the mice made antibody to human IgG. At 1 year of age most of the male mice had insulitis, although none of the male or female mice had become spontaneously diabetic. Two thirds of animals injected with cyclophosphamide at 16 months became diabetic within 3 weeks. The results confirm that treatment with CD4 antibody in the first 6 months suffices to reduce the incidence of diabetes in NOD mice. The treatment does not prevent the subsequent development of insulitis in injected mice and does not prevent the accumulation of cells capable of causing overt diabetes after cyclophosphamide injection.     

Protection of mice against Trypanosoma cruzi by immunization with paraflagellar rod proteins requires T cell, but not B cell, function

Previous studies have shown that immunization of mice with the paraflagellar rod proteins (PAR) of Trypanosoma cruzi induces an immune response capable of protecting mice against an otherwise lethal challenge with this parasite. Herein, we define immunologic responses that do or do not play a critical role in PAR-mediated protection. Firstly, PAR-immunized Ab-deficient (muMT) strain mice survived an otherwise lethal T. cruzi challenge, indicating that a B cell response is not required for PAR-induced immunity. However, beta2m -/- mice, which are severely deficient in MHC class I and TCR alphabeta+ CD8+ CD4- T cells, did not survive challenge infection following PAR immunization, indicating that MHC class I/CD8+ T cell function is necessary for protection induced by PAR immunization. Surprisingly, PAR-immunized mice depleted of CD4+ T cells survived a T. cruzi challenge for >84 days postinfection while maintaining a parasitemia that is generally thought to be lethal (i.e., >10(6) trypomastigotes/ml), thus associating CD4+ T cell function with the process of parasite clearance. Consistent with this association, CD4+ T cells from PAR-immunized mice released INF-gamma and stimulated T. cruzi-infected macrophages to release nitric oxide. The importance of IFN-gamma in PAR-induced protective immunity is further indicated by the observation that PAR-immunized INF-gamma knockout mice developed an extremely high parasitemia and did not survive a challenge infection. Thus, while Ab-mediated immune mechanisms are not required for protection induced by PAR immunization, T cell responses are necessary for both elimination of bloodstream parasites and survival.     

Persistence of maternal antibody in infants beyond 12 months: mechanism of measles vaccine failure

A serologic study was made in 34 children immunized against measles at the age of 12 months. Using a sensitive virus neutralization test, it was found that many of the children had pre-existing maternal antibody to measles virus. Children with high pre-existing antibody titers failed to seroconvert. Children with lower pre-existing antibody titers seroconverted, but the resulting antibody titer was significantly lower than in children without pre-existing antibody titer. The results of this study demonstrate a probably mechanism for measles vaccine failure in 12-month-old children and support the recommendation of the Public Health Service Advisory Committee on Immunization Practices to postpone measles vaccination to 15 months of age.     

Protective efficacy of a dengue 2 DNA vaccine in mice and the effect of CpG immuno-stimulatory motifs on antibody responses

A recently described DNA vaccine for dengue (DEN) type 2 was shown to elicit high levels of neutralizing antibodies in mice. The vaccine candidate consists of the PreM and 92% of the envelope genes of DEN 2 New Guinea C strain. We further evaluated this DNA vaccine candidate by examining the effect of immuno-stimulatory CpG DNA motifs on antibody response and by studying the protective efficacy of the vaccine. The results showed that CpG motifs present in pUC 19 significantly improved the antibody response to a suboptimal dose of 3.1 micrograms of the DEN DNA vaccine. In a lethal mouse intracerebral challenge model, the vaccine provided a significant level of protection. Sixty percent of the mice immunized with the DEN DNA vaccine plus pUC 19 survived the challenge compared to only 10% in the control group that received vector plus pUC. These studies illustrate that nucleic acid immunization is a viable approach to developing a DEN vaccine and that immuno-stimulatory CpG DNA motifs can be used to lower the minimum dose required to produce an antibody response.     

Suppression of developmental retinal cell death but not of photoreceptor degeneration in Bax-deficient mice

PURPOSE: Bax, a member of the Bcl2 family of cell death regulators, induces cell death by promoting the induction of apoptosis. Bax-deficient mice were examined in this study to determine whether Bax is required for cell death in the developing retina and for pathologic apoptotic photoreceptor degeneration resulting from the rd mutation. METHODS: Retinas from Bax-deficient mice and their wild-type siblings were harvested at postnatal day (P) 7 and processed for TdT-dUTP terminal nick-end labeling (TUNEL) staining, and the number of nuclei containing fragmented DNA were counted. Adult retinas and optic nerves were processed for plastic-embedded 1-microm sections, and the cross-sectional area was determined. The mutant Bax allele was outbred onto the C3H mouse strain, which carries the rd allele. Retinas from these animals were examined histologically at P21 after most of the photoreceptor cell death had occurred. RESULTS: At P7, around the time of peak cell death in the inner nuclear layer (INL), significantly fewer neurons in INL and ganglion cell layer (GCL) were TUNEL positive in Bax-deficient mice than in their wild-type siblings. In adult Bax-deficient mice, the cross-sectional area of the optic nerve was approximately 50% larger than in wild-type siblings, and the total number of retinal ganglion cell axons was increased to 226%. The INL of Bax-deficient mice was thicker than normal. The Bax genotype did not affect the thickness or histologic appearance of the outer nuclear layer in retinas of mice with wild-type or mutant rd alleles. CONCLUSIONS: In the absence of the expression of the Bax gene, there is a profound increase in the survival of retinal ganglion cells that lasts into adulthood. Similarly, death of INL cells is diminished but not completely abolished. The absence of Bax does not, however, protect photoreceptors from naturally occurring cell death or degeneration induced by the rd mutation. This shows that Bax is involved to a variable degree in the control of developmental cell death in the retina and that not all apoptotic retinal cell deaths are controlled by Bax.     

The immune response to soluble D10 TCR: analysis of antibody and T cell responses

Interactions between short single-stranded DNA ligands and fluorescent DNA indicator dyes were used to investigate binding selectivity of the ligands. Conformational differences among four DNA ligands of different sequence and structure, including two that form a G-quartet and two that do not, were confirmed by circular dichroism spectroscopy. Their interactions with indicator dyes YO-pro-1 iodide (YO) and YOYO-1 iodide (YOYO) were probed using measurements of dye absorbance; induced circular dichroism; and fluorescence spectra, anisotropy, and lifetime. Equilibrium binding constants and stoichiometry were determined as well. Results indicate significant differences among the dye interactions and binding stoichiometries of the four ligands. One of the G-quartet forming ligands, a 20-mer of sequence 5'-GGTTTTGGTTTTGGTTTTGG-3', shows distinctly different interactions from the other three ligands, all of which are 15-mers. These studies illustrate the importance of sequence and conformation in determining the binding interactions of short single-stranded DNA.     

Transgenic expression of Fas in T cells blocks lymphoproliferation but not autoimmune disease in MRL-lpr mice

Fas is a member of the TNF receptor family. Binding of Fas ligand to Fas induces apoptosis in Fas-bearing cells. Fas is expressed in various cells, including thymocytes, peripheral T cells, and activated B cells. The mouse lpr mutation is a loss of function mutation of Fas. MRL-lpr/lpr mice develop lymphadenopathy and splenomegaly, and produce multiple autoantibodies, which results in autoimmune disease. In this report, we describe the establishment of a line of Fas transgenic MRL-lpr mice in which mouse Fas cDNA was expressed using the T cell-specific murine lck promoter. The transgenic mice expressed functional Fas in thymocytes and peripheral T cells, but not in B cells. The transgenic mice did not accumulate abnormal T cells (Thy-1+ B220+), but still accumulated B cells (Thy-1- B220+); they produced a large quantity of Igs (IgG1 and IgG2a), including anti-DNA Abs, and developed glomerulonephritis. These results suggest that autoreactive or activated B cells must be killed through Fas expressed in the B cells by the Fas ligand expressed in activated T cells.     

CD4+ T cell responses in mice lacking MHC class II molecules specifically on B cells

The role of B lymphocytes in initiating and maintaining a CD4+ T cell response has been examined using a variety of strategies, but remains controversial because of weaknesses inherent to each of the approaches. Here, we address this issue by measuring CD4+ T cell priming both in mutant mice devoid of B cells and in chimeric animals lacking major histocompatibility complex class II molecules specifically on B cells. We find that peptide and some protein antigens do not require B cells expressing class II molecules, nor B cells themselves, to efficiently prime. This could be demonstrated by the usual lymph node proliferation assay, a rather indirect in vitro measure of priming, and by a direct ex vivo assay of population expansion and activation marker expression. Interestingly, one protein antigen, conalbumin, could not prime in the absence of B cells, but could in the presence of B cells devoid of class II molecules. This finding constrains the possible mechanisms whereby B lymphocytes contribute to the initiation of a CD4+ T cell response, arguing against the importance of surface immunoglobulin-mediated antigen presentation by B cells.     

Optimization of vaccine responses in early life: the role of delivery systems and immunomodulators

Pooled donor fibrin with an ultimate fibrinogen concentration of 60 mg/ml was used to study its effect on wound healing of surgically created ulcers in a rabbit ear. Water soluble polymer (PEG Mw = 20 KD) beads of 100-150 microns were added (12% by volume) to the fibrinogen to obtain a porous and rough structure. Five 6 mm-diameter ulcers to the depth of bare cartilage were created on each rabbit ear. There were two periods of study (4 and 8 days), with 15 ulcers in each time period, 5 of which were treated with a modified fibrin scaffold, 5 with a non-modified fibrin scaffold, and 5 served as control ulcers. The ulcer sites were subjected to routine histological processing and histomorphometrical quantification. Data analysis revealed significant increases in volume fraction of fibroblast and number of blood vessels in the modified fibrin scaffold treated ulcers over control and non-modified fibrin scaffold treated groups.     

Influence of inoculation site of combined oil-adjuvanted vaccine on the antibody response in chickens

In this study, we evaluated the relationship between resistance to insulin-mediated glucose disposal and hematocrit (Hct) and hemoglobin (Hgb) concentrations in 150 normal, healthy volunteers: 100 men and 50 women. Insulin resistance was defined as the steady-state plasma glucose (SSPG) concentration at the end of a 180-minute infusion of somatostatin, insulin, and glucose. Since the steady-state plasma insulin (SSPI) concentrations are similar in all individuals, the SSPG concentrations provide a direct measure of insulin resistance: the higher the SSPG, the more insulin-resistant the subject. The results indicated that SSPG was significantly (P < .001) related to Hct and Hgb in both men and women, with correlation coefficients (r) ranging from 0.38 to 0.43. A series of other variables were also related to Hct and Hgb, including blood pressure, plasma glucose and insulin responses to oral glucose, and plasma triglyceride and high-density lipoprotein (HDL) concentrations. When multiple regression analysis was used to evaluate these relationships, the only variables that were consistently found to be associated with Hct and Hgb were insulin resistance and plasma insulin response to oral glucose. Thus, these results suggest that Hct and Hgb concentrations be added to the cluster of variables related to insulin resistance and compensatory hyperinsulinemia.     

Treatment with anti-interleukin-10 monoclonal antibody enhances early resistance to but impairs complete clearance of Listeria monocytogenes infection in mice

Mice that received an anti-interleukin-10 (anti-IL-10) neutralizing monoclonal antibody (MAb) (SXC-1) prior to infection with Listeria monocytogenes initially demonstrated resistance to the infection, as indicated by reduced recovery of L. monocytogenes from their spleens and livers during the first 5 days after challenge. Anti-IL-10 MAb-treated mice then demonstrated reduced resistance during the later stage of infection, as indicated by persistent infection with L. monocytogenes in their livers 11 days after challenge. Aspartate aminotransferase (AST) levels (a measure of liver damage) in the sera of control mice increased between 1 and 5 days after challenge, while anti-IL-10 MAb-treated mice maintained lower AST levels. At 7 days after challenge, AST levels in the sera of control mice decreased as the numbers of organisms declined. In contrast, AST levels increased as the infections persisted in anti-IL-10 MAb-treated mice. The AST levels in serum reflected liver histopathology as anti-IL-10 MAb-treated mice exhibited fewer granulomatous lesions and less necrosis of liver tissue than the control mice during the first 5 days after challenge. Anti-IL-10 MAb treatment altered the expression of inflammatory cytokine mRNAs during L. monocytogenes infection. Control MAb-treated mice exhibited increased expression of tumor necrosis factor alpha and granulocyte-macrophage colony-stimulating factor mRNA in their lives during L. monocytogenes infection, but this increase did not occur in anti-IL-10 MAb-treated mice. Gamma interferon mRNA expression in the livers of the control MAb-treated mice was increased between 1 and 5 days after L. monocytogenes challenge and then decreased at 7 days after challenge. In contrast, gamma interferon mRNA expression in the livers of anti-IL-10 MAb-treated mice was not decreased until 7 days after challenge. These results indicate that endogenous IL-10 has both beneficial and detrimental effects on the host response to L. monocytogenes infection in mice.