Two Vaccines for Staphylococcus aureus Induce a B-Cell-Mediated Immune Response.

Staphylococcus aureus causes severe disease in humans for which no licensed vaccine exists. A novel S. aureus vaccine (SA4Ag) is in development, targeting the capsular polysaccharides (CPs) and two virulence-associated surface proteins. Vaccine-elicited antibody responses to CPs are efficacious against serious infection by other encapsulated bacteria. Studies of natural S. aureus infection have also shown a role for TH17 and/or TH1 responses in protection. Single-antigen vaccines, including CPs, have not been effective against S. aureus; a multiantigen vaccine approach is likely required. However, the impact of addition of protein antigens on the immune response to CPs has not been studied. Here, the immune response induced by a bivalent CP conjugate vaccine (to model the established mechanism of action of vaccine-induced protection against Gram-positive pathogens) was compared to the response induced by SA4Ag, which contains both CP conjugates and protein antigens, in cynomolgus macaques. Microengraving, flow cytometry, opsonophagocytic assays, and Luminex technology were used to analyze the B-cell, T-cell, functional antibody, and innate immune responses. Both the bivalent CP vaccine and SA4Ag induced cytokine production from naive cells and antigen-specific memory B-cell and functional antibody responses. Increases in levels of circulating, activated T cells were not apparent following vaccination, nor was a TH17 or TH1 response evident. However, our data are consistent with a vaccine-induced recruitment of T follicular helper (TFH) cells to lymph nodes. Collectively, these data suggest that the response to SA4Ag is primarily mediated by B cells and antibodies that abrogate important S. aureus virulence mechanisms.IMPORTANCEStaphylococcus aureus causes severe disease in humans for which no licensed vaccine exists. A novel vaccine is in development that targets multiple elements of the bacteria since single-component vaccines have not shown efficacy to date. How these multiple components alter the immune response raised by the vaccine is not well studied. We found that the addition of two protein components did not alter substantially the antibody responses raised with respect to function or mobilization of B cells. There was also not a substantial change in the activity of T cells, another part of the adaptive response. This study showed that protection by this vaccine may be mediated primarily by antibody protection.

Longitudinal multiparameter single-cell analysis of macaques immunized with pneumococcal protein-conjugated or unconjugated polysaccharide vaccines reveals distinct antigen specific memory B cell repertoires.

BACKGROUND: The efficacy of protein-conjugated pneumococcal polysaccharide vaccines has been well characterized for children. The level of protection conferred by unconjugated polysaccharide vaccines remains less clear, particularly for elderly individuals who have had prior antigenic experience through immunization with unconjugated polysaccharide vaccines or natural exposure to Streptococcus pneumoniae. METHODS: We compared the magnitude, diversity and genetic biases of antigen-specific memory B cells in two groups of adult cynomolgus macaques that were immunized with a 7-valent conjugated vaccine and boosted after five years with either a 13-valent pneumococcal polysaccharide conjugate vaccine (13vPnC) or a 23-valent unconjugated pneumococcal polysaccharide vaccine (23vPS) using microengraving (a single-cell analysis method) and single-cell RT-PCR. RESULTS: Seven days after boosting, the mean frequency of antigen-specific memory B cells was significantly increased in macaques vaccinated with 13vPnC compared to those receiving 23vPS. The 13vPnC-vaccinated macaques also exhibited a more even distribution of antibody specificities to four polysaccharides in the vaccine (PS4, 6B, 14, 23F) that were examined. However, single-cell analysis of the antibody variable region sequences from antigen-specific B cells elicited by unconjugated and conjugated vaccines indicated that both the germline gene segments forming the heavy chains and the average lengths of the Complementary Determining Region 3 (CDR3) were similar. CONCLUSIONS: Our results confirm that distinctive differences can manifest between antigen-specific memory B cell repertoires in nonhuman primates immunized with conjugated and unconjugated pneumococcal polysaccharide vaccines. The study also supports the notion that the conjugated vaccines have a favorable profile in terms of both the frequency and breadth of the anamnestic response among antigen-specific memory B cells.

Flt3 Ligand Is Essential for Survival and Protective Immune Responses during Toxoplasmosis.

Dendritic cells (DCs) are critical for resistance to Toxoplasma gondii, and infection with this pathogen leads to increased numbers of DCs at local sites of parasite replication and in secondary lymphoid organs, but the factors that regulate this expansion are poorly understood. The cytokine Flt3 ligand (Flt3L) is critical for the generation and maintenance of DCs, and Flt3L(-/-) mice were found to be highly susceptible to acute toxoplasmosis. This phenotype correlated with decreased production of IL-12 and IFN-γ, as well as impaired NK cell responses. Surprisingly, despite low basal numbers of DCs, Flt3L(-/-) mice infected with T. gondii displayed an expansion of CD8α(+) and CD11b(lo)CD8α(-) DCs. Infection also induced an expansion of parasite-specific CD4(+) and CD8(+) T cells in Flt3L(-/-) mice; however, these cells were reduced in number and displayed impaired ability to produce IFN-γ relative to wild-type controls. Exogenous IL-12 treatment partially restored NK and T cell responses in Flt3L(-/-) mice, as well as acute resistance; however, these mice eventually succumbed to toxoplasmic encephalitis, despite the presence of large numbers of DCs and T cells in the brain. These results highlight the importance of Flt3L for resistance to toxoplasmosis and demonstrate the existence of Flt3L-independent pathways that can mediate infection-induced expansion of DCs and T cell priming.

Parasite fate and involvement of infected cells in the induction of CD4+ and CD8+ T cell responses to Toxoplasma gondii.

During infection with the intracellular parasite Toxoplasma gondii, the presentation of parasite-derived antigens to CD4+ and CD8+ T cells is essential for long-term resistance to this pathogen. Fundamental questions remain regarding the roles of phagocytosis and active invasion in the events that lead to the processing and presentation of parasite antigens. To understand the most proximal events in this process, an attenuated non-replicating strain of T. gondii (the cpsII strain) was combined with a cytometry-based approach to distinguish active invasion from phagocytic uptake. In vivo studies revealed that T. gondii disproportionately infected dendritic cells and macrophages, and that infected dendritic cells and macrophages displayed an activated phenotype characterized by enhanced levels of CD86 compared to cells that had phagocytosed the parasite, thus suggesting a role for these cells in priming naïve T cells. Indeed, dendritic cells were required for optimal CD4+ and CD8+ T cell responses, and the phagocytosis of heat-killed or invasion-blocked parasites was not sufficient to induce T cell responses. Rather, the selective transfer of cpsII-infected dendritic cells or macrophages (but not those that had phagocytosed the parasite) to naïve mice potently induced CD4+ and CD8+ T cell responses, and conferred protection against challenge with virulent T. gondii. Collectively, these results point toward a critical role for actively infected host cells in initiating T. gondii-specific CD4+ and CD8+ T cell responses.

A host GPCR signaling network required for the cytolysis of infected cells facilitates release of apicomplexan parasites.

Following intracellular replication, the apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii cause host cell cytolysis to facilitate parasite release and disease progression. Parasite exit from infected cells requires the interplay of parasite-derived proteins and host actin cytoskeletal changes; however, the host proteins underlying these changes remain obscure. We report the identification of a Gα(q)-coupled host-signaling cascade required for the egress of both P. falciparum and T. gondii. Gα(q)-coupled signaling results in protein kinase C (PKC)-mediated loss of the host cytoskeletal protein adducin and weakening of the cellular cytoskeleton. This cytoskeletal compromise induces catastrophic Ca(2+) influx mediated by the mechanosensitive cation channel TRPC6, which activates host calpain that proteolyzes the host cytoskeleton allowing parasite release. Reinforcing the feasibility of targeting host proteins as an antiparasitic strategy, mammalian PKC inhibitors demonstrated activity in murine models of malaria and toxoplasmosis. Importantly, an orally bioavailable PKC inhibitor prolonged survival in an experimental cerebral malaria model.

Immune response and immunopathology during toxoplasmosis.

Toxoplasma gondii is a protozoan parasite of medical and veterinary significance that is able to infect any warm-blooded vertebrate host. In addition to its importance to public health, several inherent features of the biology of T. gondii have made it an important model organism to study host-pathogen interactions. One factor is the genetic tractability of the parasite, which allows studies on the microbial factors that affect virulence and allows the development of tools that facilitate immune studies. Additionally, mice are natural hosts for T. gondii, and the availability of numerous reagents to study the murine immune system makes this an ideal experimental system to understand the functions of cytokines and effector mechanisms involved in immunity to intracellular microorganisms. In this article, we will review current knowledge of the innate and adaptive immune responses required for resistance to toxoplasmosis, the events that lead to the development of immunopathology, and the natural regulatory mechanisms that limit excessive inflammation during this infection.

Guanylate-binding proteins: niche recruiters for antimicrobial effectors.

There are fundamental questions regarding how IFN-γ activates host cells to eliminate intracellular pathogens. In this issue of Immunity, Yamamoto et al. (2012) demonstrate a critical role for the p65 guanylate-binding proteins (GBPs) in this process during infection with Toxoplasma gondii.

Interleukin-27 priming of T cells controls IL-17 production in trans via induction of the ligand PD-L1.

Interleukin-27 (IL-27) is a key immunosuppressive cytokine that counters T helper 17 (Th17) cell-mediated pathology. To identify mechanisms by which IL-27 might exert its immunosuppressive effect, we analyzed genes in T cells rapidly induced by IL-27. We found that IL-27 priming of naive T cells upregulated expression of programmed death ligand 1 (PD-L1) in a signal transducer and activator of transcription 1 (STAT1)-dependent manner. When cocultured with naive CD4(+) T cells, IL-27-primed T cells inhibited the differentiation of Th17 cells in trans through a PD-1-PD-L1 interaction. In vivo, coadministration of naive TCR transgenic T cells (2D2 T cells) with IL-27-primed T cells expressing PD-L1 inhibited the development of Th17 cells and protected from severe autoimmune encephalomyelitis. Thus, these data identify a suppressive activity of IL-27, by which CD4(+) T cells can restrict differentiation of Th17 cells in trans.

Role of the NF-κB transcription factor c-Rel in the generation of CD8+ T-cell responses to Toxoplasma gondii.

The nuclear factor κB transcription factor c-Rel is exclusively expressed in immune cells and plays a role in numerous cellular functions including proliferation, survival and production of chemokines and cytokines. c-Rel has also been implicated in the regulation of multiple genes involved in innate and adaptive immune responses to the intracellular protozoan parasite Toxoplasma gondii, in particular IL-12. To better understand how this transcription factor controls the CD8(+) T-cell response to this organism, wild-type (WT) and c-Rel(-/-) mice were challenged with a replication-deficient strain of T. gondii that expresses the model antigen ovalbumin (OVA). These studies revealed that c-Rel was required for optimal primary expansion of OVA-specific CD8(+) T cells and that immunized c-Rel-deficient mice were susceptible to challenge with a virulent strain of T. gondii. However, when c-Rel(-/-) cells specific for OVA were adoptively transferred into a WT recipient, or c-Rel(-/-) mice were treated with IL-12 at the time of immunization, there was no apparent proliferative defect. Surprisingly, upon secondary challenge, antigen-specific CD8(+) T cells in c-Rel(-/-) mice expanded to a much greater degree in terms of frequency as well as numbers when compared with WT mice. Despite this, the cytokine responses of c-Rel(-/-) mice remained defective, consistent with their susceptibility to secondary challenge. Together, these results indicate that in this infection model, the major influence of c-Rel in generation of CD8(+) T-cell responses is through its regulation of the inflammatory environment, rather than playing a substantial T-cell-intrinsic role.

Virulence of Toxoplasma gondii is associated with distinct dendritic cell responses and reduced numbers of activated CD8+ T cells.

The Toxoplasma gondii population consists of multiple strains, defined by genotype and virulence. Previous studies have established that protective immunity to this organism is mediated by IL-12, which drives T cells to produce IFN-gamma. Paradoxically, although type I and type II strains of T. gondii both induce IL-12 and IFN-gamma in the mouse, type I parasites are lethal, whereas type II strains establish chronic infection. The cellular basis for these strain-dependent differences remains unclear. To better understand these events, the CD8(+) T cell and dendritic cell (DC) responses to transgenic, OVA-expressing type I RH (RH OVA) and type II Prugniuad (Pru OVA) parasites were examined. Pru OVA-infected mice developed a robust DC response at the site of infection and the draining lymph node and generated a population of endogenous OVA-specific CD8(+) T cells. In contrast, RH OVA-infected mice had fewer DCs and OVA-specific CD8(+) T cells. RH OVA-infected mice given preactivated OVA-specific CD8(+) T cells were protected, suggesting that reduced DC-derived signals contributed to the low OVA-specific CD8(+) T cell numbers observed during type I infection. Indeed, DC depletion prior to Pru OVA infection resulted in a failure to generate activated OVA-specific CD8(+) T cells, and IL-12p70 treatment during RH OVA infection modestly increased the number of Ag-specific cells. Together, these data are consistent with a model of immunity to T. gondii in which strain-dependent DC responses shape the generation of Ag-specific CD8(+) T cells and determine the outcome of infection.

African trypanosomes contain 5-methylcytosine in nuclear DNA.

It is currently unclear if there are modified DNA bases in Trypanosoma brucei other than J-base. We identify herein a cytosine-5 DNA methyltransferase gene and report the presence and location of 5-methylcytosine in genomic DNA. Our data demonstrate that African trypanosomes contain a functional cytosine DNA methylation pathway.