Vitamin Supplementation at the Time of Immunization with a Cold-Adapted Influenza Virus Vaccine Corrects Poor Mucosal Antibody Responses in Mice Deficient for Vitamins A and D.

Vitamin A and D deficiencies and insufficiencies are prevalent worldwide in developed and developing countries. Vitamin metabolites are functionally intertwined in that they are high-affinity ligands for related receptors of the nuclear receptor superfamily. The effects of vitamin A deficiencies (VAD) on antibody responses to respiratory virus vaccines have already been demonstrated. Of particular concern was the reduction in IgA, a first line of defense against pathogens in the respiratory tract. Here, we describe the individual and combined effects of vitamin A and D deficiencies in mice immunized with an attenuated influenza virus vaccine. Relative to VAD, vitamin D deficiency (VDD) had a limited effect, but double deficiencies for vitamins A and D (VAD+VDD) further reduced antibody responses in the respiratory tract. The administration of supplemental vitamins A and D to VAD+VDD mice at the time of vaccination restored responses in a dose-dependent manner. Results suggest that vitamin supplementation programs may be beneficial in a clinical setting to promote healthy immune responses to respiratory virus vaccines in vitamin-deficient individuals.

Oral retinyl palmitate or retinoic acid corrects mucosal IgA responses toward an intranasal influenza virus vaccine in vitamin A deficient mice.

Vitamin A deficiency (VAD) is a leading cause of pediatric morbidity and mortality due to infectious diseases. Recent pre-clinical studies have revealed that VAD impairs mucosal IgA-producing antibody forming cell (AFC) responses toward a paramyxovirus vaccine in the upper respiratory tract (URT), thus impeding a first line of defense at the pathogen's point-of-entry. The studies described here tested the hypothesis that VAD may also impair immune responses after FluMist vaccinations. Results show that (i) IgA-producing antibody forming cells (AFCs) are significantly reduced following FluMist vaccination in VAD mice, and (ii) oral doses of either retinyl palmitate or retinoic acid administered on days 0, 3, and 7 relative to vaccination rescue the response. Data encourage the conduct of clinical studies to determine if there are FluMist vaccine weaknesses in human VAD populations and to test corrective supplementation strategies. Improvements in vaccine efficacy may ultimately reduce the morbidity and mortality caused by influenza virus worldwide.

Robust IgA and IgG-producing antibody forming cells in the diffuse-NALT and lungs of Sendai virus-vaccinated cotton rats associate with rapid protection against human parainfluenza virus-type 1.

Sendai virus (SeV), a natural mouse pathogen, shows considerable promise as a candidate vaccine for human parainfluenza virus-type 1 (hPIV-1), and also as a vaccine vector for other serious pathogens of infants including respiratory syncytial virus (RSV). In an effort to define correlates of immunity, we examined the virus-specific serum antibody of cotton rats inoculated intranasally (I.N.) with SeV. Virus-specific antibody forming cells (AFCs) were also measured in the bone marrow, because these are considered responsible for durable serum antibody levels in other viral systems. Results showed that a single SeV inoculation was sufficient to induce virus-specific serum antibodies and bone marrow-resident AFCs that persisted for as many as 8 months post-vaccination. Given that the predominant SeV-specific serum antibody isotype was IgG, an isotype that traffics poorly to the upper respiratory tract (URT), we asked if local nasal and lung-associated antibodies and AFCs were also present. Studies showed that: (i) SeV-specific antibodies appeared in the URT and lower respiratory tract (LRT) within 7 days after immunization, (ii) corresponding AFCs were present in the diffuse-NALT (d-NALT) and lung, (iii) AFCs in the d-NALT and lung peaked at approximately 6 weeks and persisted for the lifetime of the animal, reaching a level exceeding that of the bone marrow by an order of magnitude, (iv) IgA was the dominant isotype among AFCs in the d-NALT and lung at 4-weeks post-vaccination and thereafter, and (v) antibody and AFC responses associated with the prevention of lung infection when animals were challenged with hPIV-1 just 1 week after vaccination.

Identification of MHC class II-associated peptides that promote the presentation of toxic shock syndrome toxin-1 to T cells.

Previous studies have shown that the DM-deficient cell line, T2-I-A(b), is very inefficient at presenting toxic shock syndrome toxin 1 (TSST-1) to T cells, suggesting that I-A(b)-associated peptides play an essential role in the presentation of this superantigen. Consistent with this, the loading of an I-A(b)-binding peptide, staphylococcal enterotoxin B 121-136, onto T2-I-A(b) cells enhanced TSST-1 presentation >1000-fold. However, despite extensive screening, no other peptides have been identified that significantly promote TSST-1 presentation. In addition, the peptide effect on TSST-1 presentation has been demonstrated only in the context of the tumor cell line T2-I-A(b). Here we show that peptides that do not promote TSST-1 presentation can be converted into "promoting" peptides by the progressive truncation of C-terminal residues. These studies result in the identification of two peptides derived from IgGV heavy chain and I-Ealpha proteins that are extremely strong promoters of TSST-1 presentation (47,500- and 12,000-fold, respectively). We have also developed a system to examine the role of MHC class II-associated peptides in superantigen presentation using splenic APC taken directly ex vivo. The data confirmed that the length of the MHC class II-bound peptide plays a critical role in the presentation of TSST-1 by splenic APC and showed that different subpopulations of APC are equally peptide dependent in TSST-1 presentation. Finally, we demonstrated that the presentation of staphylococcal enterotoxin A, like TSST-1, is peptide dependent, whereas staphylococcal enterotoxin B presentation is peptide independent.

Control of gammaherpesvirus latency by latent antigen-specific CD8(+) T cells.

The contribution of the latent antigen-specific CD8(+) T cell response to the control of gammaherpesvirus latency is currently obscure. Some latent antigens induce potent T cell responses, but little is known about their induction or the role they play during the establishment of latency. Here we used the murine gammaherpesvirus system to examine the expression of the latency-associated M2 gene during latency and the induction of the CD8(+) T cell response to this protein. M2, in contrast to the M3 latency-associated antigen, was expressed at day 14 after infection but was undetectable during long-term latency. The induction of the M2(91-99)/K(d) CD8(+) T cell response was B cell dependent, transient, and apparently induced by the rapid increase in latently infected cells around day 14 after intranasal infection. These kinetics were consistent with a role in controlling the initial "burst" of latently infected cells. In support of this hypothesis, adoptive transfer of an M2-specific CD8(+) T cell line reduced the initial load of latently infected cells, although not the long-term load. These data represent the first description of a latent antigen-specific immune response in this model, and suggest that vaccination with latent antigens such as M2 may be capable of modulating latent gammaherpesvirus infection.

Functionally heterogeneous CD8(+) T-cell memory is induced by Sendai virus infection of mice.

It has recently been established that memory CD8(+) T cells induced by viral infection are maintained at unexpectedly high frequencies in the spleen. While it has been established that these memory cells are phenotypically heterogeneous, relatively little is known about the functional status of these cells. Here we investigated the proliferative potential of CD8(+) memory T cells induced by Sendai virus infection. High frequencies of CD8(+) T cells specific for both dominant and subdominant Sendai virus epitopes persisted for many weeks after primary infection, and these cells were heterogeneous with respect to CD62L expression (approximately 20% CD62L(hi) and 80% CD62L(lo)). Reactivation of these cells with the antigenic peptide in vitro induced strong proliferation of antigen-specific CD8(+) T cells. However, approximately 20% of the cells failed to proliferate in vitro in response to a cognate peptide but nevertheless differentiated into effector cells and acquired full cytotoxic potential. These cells also expressed high levels of CD62L (in marked contrast to the CD62L(lo) status of the proliferating cells in the culture). Direct isolation of CD62L(hi) and CD62L(lo) CD8(+) T cells from memory mice confirmed the correlation of this marker with proliferative potential. Taken together, these data demonstrate that Sendai virus infection induces high frequencies of memory CD8(+) T cells that are highly heterogeneous in terms of both their phenotype and their proliferative potential.

Long-term CD8+ T cell memory to Sendai virus elicited by DNA vaccination.

The capacity of DNA vaccines to prime CD8+ T cells makes them excellent candidates for vaccines that are designed to emphasize cellular immunity. However, the long-term stability of CD8+ T cell memory induced by DNA vaccination is poorly characterized. Here, the quality of CD8+ T cell recall responses in mice was investigated more than 1 year after DNA vaccination with the Sendai virus nucleoprotein gene. Cytotoxic T lymphocyte (CTL) activity specific for both dominant and subdominant epitopes could be recalled readily 1 year after vaccination and the frequencies of CTL precursors specific for both of these epitopes were relatively high. These CTL responded strongly to subsequent Sendai virus infection in terms of their ability to migrate to the lung and to differentiate into effector cells. In addition, the recall response to virus infection, as determined by CTL activity in the lungs and IFN-gamma responses in the spleen, was both faster and greater in magnitude than that in control-immunized mice. Significantly, virus titres were reduced at least 100-fold in the lungs of mice that were immunized more than 1 year before infection, as compared with control mice. These data demonstrate that CD8+ T cell memory elicited by DNA vaccination is functionally relevant and persists for at least 1 year.

Thymic lymphoproliferative disease after successful correction of CD40 ligand deficiency by gene transfer in mice.

Inherited deficiency of the CD40 ligand (X-linked hyper-IgM syndrome) is characterized by failure of immunoglobulin isotype switching and severe defects of cell-mediated immunity. To test the potential for gene transfer therapy to correct this disorder, we transduced murine bone marrow or thymic cells with a retroviral vector containing the cDNA for the murine CD40 ligand (CD40L) and injected them into CD40L-/- mice. Even low-level, constitutive expression of the transgene stimulated humoral and cellular immune functions in these mice. With extended follow-up, however, 12 of 19 treated mice developed T-lymphoproliferative disorders, ranging from polyclonal increases of lymphoblasts to overt monoclonal T-lymphoblastic lymphomas that involved multiple organs. Our findings show that constitutive (rather than tightly regulated), low-level expression of CD40L can produce abnormal proliferative responses in developing T lymphocytes, apparently through aberrant interaction between CD40L+ and TCRalphabeta+CD40+ thymocytes. Current methods of gene therapy may prove inappropriate for disorders involving highly regulated genes in essential positions in proliferative cascades.