There is considerable interest in pneumococcal protein antigens capable of inducing serotype-independent immunoprotection and of improving, thereby, existing vaccines. We report here on the immunogenic properties of a novel surface antigen encoded by ORF spr1875 in the R6 strain genome. An antigenic fragment encoded by spr1875, designated R4, was identified using a Streptococcus pneumoniae phage displayed genomic library after selection with a human convalescent serum. Immunofluorescence analysis with anti-R4 antisera showed that Spr1875 was expressed on the surface of strains belonging to different serotypes. Moreover, the gene was present with little sequence variability in 27 different pneumococcal strains isolated worldwide. A mutant lacking Spr1875 was considerably less virulent than the wild type D39 strain in an intravenous mouse model of infection. Moreover, immunization with the R4 recombinant fragment, but not with the whole Spr1875 protein, induced significant protection against sepsis in mice. Lack of protection after immunization with the whole protein was related to the presence of immunodominant, non-protective epitopes located outside of the R4 fragment. In conclusion, our data indicate that Spr1875 has a role in pneumococcal virulence and is immunogenic. As the R4 fragment conferred immunoprotection from experimental sepsis, selected antigenic fragments of Spr1875 may be useful for the development of a pneumococcal protein-based vaccine.

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The gamma interferon (IFN-␥) assay is widely used to measure cell-mediated immune (CMI) response for the early detection of tuberculosis infection. Processing whole-blood samples for CMI-based diagnostics is time sensitive and usually must occur within 8 h of collection to ensure optimal assay performance. In this study, we developed and tested a modified protocol, in which whole-blood samples from Mycobacterium bovis-infected cattle were diluted 1:1 in RPMI medium containing 0.3% fetal bovine serum (FBS) added or not to recombinant mouse interleukin-7 (rmIL-7) or rmIL-12, alone or in combination, and stored at 4°C. At 3 and 6 days postcollection, the diluted blood samples were adjusted to 10% FBS, dispensed into culture trays, stimulated with a bovine purified protein derivative from M. bovis, and incubated at 37°C in 5% CO 2 in air. Plasma was removed and assayed for an IFN-␥ response using bovine IFN-␥ enzyme-linked immunosorbent assay (ELISA) (Bovigam). The results were then compared with those obtained from the conventional procedure. The IFN-␥ responses of the samples stored up to 6 days postcollection in the supplemented RPMI medium were similar to those observed in the samples processed within 8 h after sampling, indicating that lymphocyte vitality and response were preserved. The addition of rmIL-7 and rmIL-12, alone or in combination, to culture medium can enhance lymphocyte survival and thus extends the time limit within which the IFN-␥ assay can be applied as a diagnostic tool in bovine tuberculosis surveillance and eradication. Bovine tuberculosis (bTB) is a chronic debilitating disease that can take years before causing clinical signs to develop. bTB eradication programs in cattle are based on the application of diagnostic tests and the culling of reactor cattle (1). The standard test for the antemortem screening of infected animals is the tuberculin skin test, which is widely used in veterinary and human medicine. Several factors can influence the specificity and sensitivity of the tuberculin intradermal test (IDT). One of its main disadvantages is that cattle may sometimes be infected by other Mycobacterium strains that produce cross-reactivity that can be easily mistaken for a positive Mycobacterium bovis infection response. In addition, it has been observed that animal age and housing can also affect test specificity (2) and that the test can turn negative in the presence of a tuberculosis infection in immunosuppressed animals. To overcome these limitations, a cell-mediated immunity (CMI) response can be determined in vitro by measuring the gamma interferon (IFN-␥) produced by memory T lymphocytes from M. bovis-infected animals. IFN-␥ in the plasma of blood cultures is measured by enzyme-linked immunosorbent assay (ELISA). The IFN-␥ assay has been shown to be a sensitive and specific test to detect tuberculosis in cattle (3) and other animal species, including sheep (4), goats (5), buffalo (6), cervids (7), and pigs (8), and it is effective for identifying infected animals at early stages of in...