Immune response to pneumococcal polysaccharides 4 and 14 in elderly and young adults. I Antibody concentrations, avidity and functional activity

Streptococcus pneumoniae is a serious worldwide pathogen and the focus of numerous vaccine development projects. Currently the most widely accepted surrogate marker for evaluating the efficacy of a given vaccine is to utilize ELISA. Measurement of antibody concentration by ELISA without reduction in cross-reactive antibodies causes an overestimation of antibody concentration and therefore protection, this is most notable in the aged, an at risk group for this infection. We compared the immune response to the pneumococcal polysaccharides (PPS) 4 and 14 of 20 young to 20 elderly adults. Pre-and post-vaccination IgG antibody concentrations and antibody avidity against PPS4 and PPS14 were measured using two different enzyme-linked immunosorbant assay (ELISA) absorption protocols. All sera were pre-absorbed with either cell-wall polysaccharide (CPS), or CPS and serotype 22F polysaccharide. Pre- and post-vaccination IgG antibody concentrations for serotype 4, but not 14, were significantly lowered with the additional absorption with serotype 22F polysaccharide in both age groups. Young and elderly demonstrated a significant increase from pre- to post-immunization antibody concentration, using either absorption method; and opsonophagocytic antibody titers in response to both PPS4 and PPS14. The correlation coefficients between ELISA and opsonophagocytic assays were improved by additional absorption with serotype 22F in response to serotype 4, but not serotype 14 in all age groups. Opsonophagocytic antibody titers in a sub-group of elderly (>77 years of age) were significantly lower than the opsonophagocytic antibody concentrations in young adults. These results suggest the importance of eliminating cross-reactive antibodies from ELISA measurements by absorption of serum and an age-related impairment in the antibody response to pneumococcal polysaccharides

Formulation of a mmaA4 Gene Deletion Mutant of Mycobacterium bovis BCG in Cationic Liposomes Significantly Enhances Protection against Tuberculosis

A new vaccination strategy is urgently needed for improved control of the global tuberculosis (TB) epidemic. Using a mouse aerosol Mycobacterium tuberculosis challenge model, we investigated the protective efficacy of a mmaA4 gene deletion mutant of Mycobacterium bovis BCG (ΔmmaA4BCG) formulated in dimethyl dioctadecyl ammonium bromide (DDA) – D(+) trehalose 6,6 dibenenate (TDB) (DDA/TDB) adjuvant. In previous studies, deletion of the mmaA4 gene was shown to reduce the suppression of IL-12 production often seen after mycobacterial infections. While the non-adjuvanted ΔmmaA4BCG strain did not protect mice substantially better than conventional BCG against a tuberculous challenge in four protection experiments, the protective responses induced by the ΔmmaA4BCG vaccine formulated in DDA/TDB adjuvant was consistently increased relative to nonadjuvanted BCG controls. Furthermore, the ΔmmaA4BCG-DDA/TDB vaccine induced significantly higher frequencies of multifunctional (MFT) CD4 T cells expressing both IFNγ and TNFα (double positive) or IFNγ, TNFα and IL-2 (triple positive) than CD4 T cells derived from mice vaccinated with BCG. These MFT cells were characterized by having higher IFNγ and TNFα median fluorescence intensity (MFI) values than monofunctional CD4 T cells. Interestingly, both BCG/adjuvant and ΔmmaA4BCG/adjuvant formulations induced significantly higher frequencies of CD4 T cells expressing TNFα and IL-2 than nonadjuvanted BCG or ΔmmaA4BCG vaccines indicating that BCG/adjuvant mixtures may be more effective at inducing central memory T cells. Importantly, when either conventional BCG or the mutant were formulated in adjuvant and administered to SCID mice or immunocompromised mice depleted of IFNγ, significantly lower vaccine-derived mycobacterial CFU were detected relative to immunodeficient mice injected with non-adjuvanted BCG. Overall, these data suggest that immunization with the ΔmmaA4BCG/adjuvant formulation may be an effective, safe, and relatively inexpensive alternative to vaccination with conventional BCG

Immune Response to Pneumococcal Polysaccharides 4 and 14 in Elderly and Young Adults: Analysis of the Variable Heavy Chain Repertoire

Streptococcus pneumoniae is a leading cause of morbidity and mortality in both developed and developing countries. The current pneumococcal polysaccharide (PPS) vaccine is highly effective in young adults; however, vaccine efficacy is dramatically decreased in the elderly population. We hypothesized that the decreased vaccine efficacy in the elderly results from altered variable gene family usage. We have characterized the immunoglobulin G gene usage of the antibody response to PPS4 and PPS14 in 20 young and 20 elderly adults. The variable heavy (V(H)) gene repertoire of human peripheral B cells was amplified by using PCR. A total of 364 heavy chain sequences with specificity for PPS4 and 305 heavy chain sequences for PPS14 were analyzed from young adults. In addition, a total of 325 sequences for PPS4 and 291 sequences for PPS14 were obtained from elderly adults. Complete sequence identity, somatic mutation frequencies, and V(H) gene usage was determined in response to PPS4 and PPS14. In all volunteers, the immune response to both polysaccharides consisted predominantly of heavy chains belonging to the V(H)3 gene family. There were significant differences in the variable gene repertoire between young and elderly adults. Somatic mutation occurred more frequently in sequences derived from young compared to elderly derived sequences. With aging, a loss of oligoclonality was noted in response to PPS4 and PPS14 compared to young adults. The observed differences in V(H) repertoire, somatic mutation, and loss of oligoclonality may contribute to decreased vaccine efficacy in the elderly

Evaluation of the anti-tuberculosis protective immunity induced by BCG strains formulated in DDA/TDB adjuvant at one month and four months post-challenge.

<p>BCGA4/Adj = <i>ΔmmaA4</i> BCG formulated in DDA/TDB adjuvant.</p><p>( ) The difference between naïve and experimental CFU.</p><p>ND – not done.</p>*<p>Significantly decreased CFU values compared to naive controls, p<0.05.</p>#<p>Significantly decreased CFU values relative to BCG, p<0.05.</p>∧<p>Significantly decreased CFU values relative to BCGA4, p<0.05.</p

Reduced BCG splenic CFU levels one month after treatment of immunocompromised mice with the adjuvanted BCG preparations.

<p>A. Mice were given two i.p. injections of anti-IFNγ mAb (black bars) two days apart before receiving a single 10⁶ CFU dose of either BCG or <i>ΔmmaA4</i>BCG with or without adjuvant. Untreated mice served as infection controls (gray bars). The antibody treatment was repeated every 10 days until the mice were sacrificed one month post-infection to quantify the splenic BCG CFU. B. SCID mice were injected with a single 10⁶ CFU dose of either BCG or <i>ΔmmaA4</i>BCG with or without adjuvant. One month after the injection, splenic bacterial burdens were determined. *Significant differences relative to adjuvanted BCG controls, <i>p</i><0.05.</p

The frequency (%) of (A) monofunctional or (B) multifunctional CD8 T cells in naïve and vaccinated mice as determined by multiparameter flow cytometry analysis.

<p>* Statistical significance relative to naives, <i>p</i><0.05.</p

Protection in C57BL/6 mice against a <i>M. tuberculsosis</i> Erdman aerosol challenge after vaccination with either BCG or the <i>ΔmmaA4</i>BCG/adjuvant formulation.

<p>Mice were challenged with <i>M. tuberculosis</i> 8 weeks after a single s.c. immunization and then were sacrificed 1, 2 or 4 months after the challenge for enumeration of CFU's in the lung. Significant CFU reduction relative to * naïve mice (<i>p</i><0.05) or ^# BCG-vaccinated controls (<i>p</i><0.05).</p

Median fluorescence intensity (MFI) of (A) IFNγ or (B) TNFα in monofunctional or multifunctional CD4 T cells.

<p>The data are presented as the mean of individual MFI values for 4–5 mice per vaccine group. * Significant differences relative to monofunctional cytokine expressing cells, <i>p</i><0.05. ^# Significant differences relative to BCG in the same T cell subset, p<0.05.</p