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ABSTRACTLive oral Salmonella entericaserovar Typhi vaccine Ty21a induces specific antibodies that cross-react against Salmonella entericaserovar Paratyphi A and Salmonella entericaserovar Paratyphi B, although their functional role in clearance remains unknown. We utilized an in vitroassay with THP-1 macrophages to compare the phagocytosis and survival of Salmonellaopsonized with heat-inactivated human sera obtained before and after vaccination with Ty21a or a live oral S. Typhi vaccine, CVD 909. Opsonization with postvaccination sera predominantly increased the phagocytosis of S. Typhi relative to the corresponding prevaccination sera, and increases were also observed with S. Paratyphi A and S. Paratyphi B, albeit of lower magnitudes. Relative to prevaccination sera, opsonization with the postvaccination sera reduced the survival inside macrophages of S. Typhi but not of S. Paratyphi A or S. Paratyphi B. Higher anti-S. Typhi O antigen (lipopolysaccharide [LPS]) IgG, but not IgA, antibody titers correlated significantly with postvaccination increases in opsonophagocytosis. No differences were observed between immunization with four doses of Ty21a or one dose of CVD 909. Ty21a and CVD 909 induced cross-reactive functional antibodies, predominantly against S. Typhi. IgG anti-LPS antibodies may be important in phagocytic clearance of these organisms. Therefore, measurement of functional antibodies might be important in assessing the immunogenicity of a new generation of typhoid and paratyphoid A vaccines. (The CVD 909 study has been registered at ClinicalTrials.gov under registration no. NCT00326443.)

ABSTRACTEnteric fever caused by Salmonella entericaserovar Paratyphi A infection has emerged as an important public health problem. Recognizing that in randomized controlled field trials oral immunization with attenuated S. entericaserovar Typhi live vaccine Ty21a conferred significant cross-protection against S. Paratyphi B but not S. Paratyphi A disease, we undertook a clinical study to ascertain whether humoral immune responses could explain the field trial results. Ty21a immunization of adult residents of Maryland elicited predominantly IgA antibody-secreting cells (ASC) that recognize S. Typhi lipopolysaccharide (LPS). Cross-reactivity to S. Paratyphi A LPS was significantly lower than that to S. Paratyphi B LPS. ASC producing IgG and IgA that bind LPS from each of these Salmonellaserovars expressed CD27 and integrin a4ß7 (gut homing), with a significant proportion coexpressing CD62L (secondary lymphoid tissue homing). No significant differences were observed in serum antibody against LPS of the different serovars. Levels of IgA B memory (BM) cells to S. Typhi LPS were significantly higher than those against S. Paratyphi A or B LPS, with no differences observed between S. Paratyphi A and B. The response of IgA BMto outer membrane proteins (OMP) from S. Typhi was significantly stronger than that to OMP of S. Paratyphi A but similar to that to OMP of S. Paratyphi B. The percentages of IgG or IgA BMresponders to LPS or OMP from these Salmonellastrains were similar. Whereas cross-reactive humoral immune responses to S. Paratyphi A or B antigens are demonstrable following Ty21a immunization, they cannot explain the efficacy data gleaned from controlled field trials.

T cells are likely to play an important role in the host defense against Salmonella enterica serovar Typhi, the causative agent of typhoid fever. We have shown that HLA-E can function as a restriction element for S. Typhi-specific CD8+T cells. Because of the potential importance of HLA-E-restricted CD8+responses in resistance to Salmonella infection, we characterized these responses and investigated their kinetics of appearance and persistence in volunteers immunized orally with the licensed attenuated Ty21a strain typhoid vaccine. Cells were obtained from volunteers before and at days 2, 4, 7, 10, 14, 28, 42, 56, 120, 180, 360, and 720 after immunization. An ex vivo multicolor staining panel including antibodies to CD107a and -b, interleukin-2, gamma interferon (IFN-), and tumor necrosis factor alpha (TNF-) was used to functionally assess memory T-cell subsets by flow cytometry. Increases in cytokine-secreting CD8+cells were observed in the T effector/memory (TEM) and CD45RA+TEM(TEMRA) subsets as early as 4 days after immunization and persisted, particularly in the TEMRAsubset, up to 2 years after immunization. The majority of HLA-E-restricted CD8+cells 28 to 56 days after immunization coexpressed CD107, IFN-, and TNF-, showing characteristic features of multifunctional T cells. In summary, the multifunctionality and longevity of the HLA-E-restricted CD8 responses observed in this study highlight their significance in adaptive immunity to S. Typhi. Finally, this is the first demonstration, in either animals or humans, of the presence of long-term multifunctional HLA-E-restricted CD8+cells after immunization.

ABSTRACTT cells are likely to play an important role in the host defense against Salmonella entericaserovar Typhi, the causative agent of typhoid fever. We have shown that HLA-E can function as a restriction element for S. Typhi-specific CD8+T cells. Because of the potential importance of HLA-E-restricted CD8+responses in resistance to Salmonellainfection, we characterized these responses and investigated their kinetics of appearance and persistence in volunteers immunized orally with the licensed attenuated Ty21a strain typhoid vaccine. Cells were obtained from volunteers before and at days 2, 4, 7, 10, 14, 28, 42, 56, 120, 180, 360, and 720 after immunization. An ex vivomulticolor staining panel including antibodies to CD107a and -b, interleukin-2, gamma interferon (IFN-?), and tumor necrosis factor alpha (TNF-a) was used to functionally assess memory T-cell subsets by flow cytometry. Increases in cytokine-secreting CD8+cells were observed in the T effector/memory (TEM) and CD45RA+TEM(TEMRA) subsets as early as 4 days after immunization and persisted, particularly in the TEMRAsubset, up to 2 years after immunization. The majority of HLA-E-restricted CD8+cells 28 to 56 days after immunization coexpressed CD107, IFN-?, and TNF-a, showing characteristic features of multifunctional T cells. In summary, the multifunctionality and longevity of the HLA-E-restricted CD8 responses observed in this study highlight their significance in adaptive immunity to S. Typhi. Finally, this is the first demonstration, in either animals or humans, of the presence of long-term multifunctional HLA-E-restricted CD8+cells after immunization.

CD8+ T cells are likely to play an important role in host defense against Salmonella enterica serovar Typhi by several effector mechanisms, including lysis of infected cells (cytotoxicity) and gamma interferon (IFN-γ) secretion. In an effort to better understand these responses, we studied the T-cell receptor (TCR) repertoire of serovar Typhi-specific CD8+ T cells in humans. To this end, we determined the TCR beta chain (V{szligbeta}) usage of CD8+ T cells from three volunteers orally immunized with Ty21a typhoid vaccine by flow cytometry using a panel of monoclonal antibodies. Although TCR V{szligbeta} usage varied among volunteers, we identified oligoclonal V{szligbeta} subset expansions in individual volunteers (V{szligbeta} 2, 5.1, 8, 17, and 22 in volunteer 1; V{szligbeta} 1, 2, 5.1, 14, 17, and 22 in volunteer 2; and V{szligbeta} 3, 8, 14, and 16 in volunteer 3). These subsets were antigen specific, as shown by cytotoxicity and IFN-γ secretion assays on V{szligbeta} sorted cells and on T-cell clones derived from these volunteers. Moreover, eight-color flow cytometric analysis showed that these clones exhibited a T effector memory phenotype (i.e., CCR7– CD27– CD45RO+ CD62L–) and coexpressed gut homing molecules (e.g., high levels of integrin α4{szligbeta}7, intermediate levels of CCR9, and low levels of CD103). In conclusion, our results show that long-term T-cell responses to serovar Typhi in Ty21a vaccinees are oligoclonal, involving multiple TCR V{szligbeta} families. Moreover, these serovar Typhi-specific CD8+ T cells bearing defined V{szligbeta} specificities are phenotypically and functionally consistent with T effector memory cells with preferential gut homing potential.

ABSTRACTCD8+T cells are likely to play an important role in host defense against Salmonella entericaserovar Typhi by several effector mechanisms, including lysis of infected cells (cytotoxicity) and gamma interferon (IFN-γ) secretion. In an effort to better understand these responses, we studied the T-cell receptor (TCR) repertoire of serovar Typhi-specific CD8+T cells in humans. To this end, we determined the TCR beta chain (Vβ) usage of CD8+T cells from three volunteers orally immunized with Ty21a typhoid vaccine by flow cytometry using a panel of monoclonal antibodies. Although TCR Vβ usage varied among volunteers, we identified oligoclonal Vβ subset expansions in individual volunteers (Vβ 2, 5.1, 8, 17, and 22 in volunteer 1; Vβ 1, 2, 5.1, 14, 17, and 22 in volunteer 2; and Vβ 3, 8, 14, and 16 in volunteer 3). These subsets were antigen specific, as shown by cytotoxicity and IFN-γ secretion assays on Vβ sorted cells and on T-cell clones derived from these volunteers. Moreover, eight-color flow cytometric analysis showed that these clones exhibited a T effector memory phenotype (i.e., CCR7−CD27−CD45RO+CD62L−) and coexpressed gut homing molecules (e.g., high levels of integrin α4β7, intermediate levels of CCR9, and low levels of CD103). In conclusion, our results show that long-term T-cell responses to serovar Typhi in Ty21a vaccinees are oligoclonal, involving multiple TCR Vβ families. Moreover, these serovar Typhi-specific CD8+T cells bearing defined Vβ specificities are phenotypically and functionally consistent with T effector memory cells with preferential gut homing potential.

ABSTRACTShigellais an important cause of diarrheal disease in young children living in developing countries. No approved vaccines are available, and the development of vaccine candidates has been hindered by the lack of firm immunological correlates of protection, among other reasons. To address this gap in knowledge, we established quantitative assays to measure Shigella-specific serum bactericidal antibody (SBA) and opsonophagocytic killing antibody (OPKA) activities and investigated their potential association with protection against disease in humans. SBA, OPKA, and Ipa-, VirG (IscA)-, and Shigella flexneri2a lipopolysaccharide-specific serum IgG titers were determined in adult volunteers who received Shigellavaccine candidate EcSf2a-2 and in unvaccinated controls, all of whom were challenged with virulent Shigella flexneri2a. Prechallenge antibody titers were compared with disease severity after challenge. SBA and OPKA, as well as IpaB- and VirG-specific IgG, significantly correlated with reduced illness. SBA and OPKA assays were also used to evaluate the immunogenicity of leading live attenuated vaccine candidates ShigellaCVD 1204 and CVD 1208S in humans. A single oral immunization with CVD 1204 or CVD 1208S resulted in SBA seroconversion rates of 71% and 47% and OPKA seroconversion rates of 57% and 35%, respectively. Higher functional antibody responses were induced by CVD 1204, which is consistent with its lower attenuation. This is the first demonstration of SBA, OPKA, and IpaB- and VirG-specific IgG levels as potential serological correlates of protection against shigellosis in humans. These results warrant further studies to establish their capacity to predict protective immunity and vaccine efficacy.