Rodolfo Pastelin-Palacios

Salmonella porins induce a sustained, lifelong specific bactericidal antibody memory response

We examined the ability of porins from Salmonella enterica serovar typhi to induce a long‐term antibody response in BALB/c mice. These porins triggered a strong lifelong production of immunoglobulin G (IgG) antibody in the absence of exogenous adjuvant. Analysis of the IgG subclasses produced during this antibody response revealed the presence of the subclasses IgG2b, IgG1, IgG2a and weak IgG3. Despite the high homology of porins, the long‐lasting anti‐S. typhi porin sera did not cross‐react with S. typhimurium. Notably, the antiporin sera showed a sustained lifelong bactericidal‐binding activity to the wild‐type S. typhi strain, whereas porin‐specific antibody titres measured by enzyme‐linked immunosorbent assay (ELISA) decreased with time. Because our porin preparations contained the outer membrane proteins C and F (OmpC and OmpF), we evaluated the individual contribution of each porin to the long‐lasting antibody response. OmpC and OmpF induced long‐lasting antibody titres, measured by ELISA, which were sustained for 300 days. In contrast, although OmpC induced sustained high bactericidal antibody titres for 300 days, postimmunization, the bactericidal antibody titre induced by OmpF was not detected at day 180. These results indicate that OmpC is the main protein responsible for the antibody‐mediated memory bactericidal response induced by porins. Taken together, our results show that porins are strong immunogens that confer lifelong specific bactericidal antibody responses in the absence of added adjuvant.

TLR2 and TLR4 signaling shapes specific antibody responses to Salmonella typhi antigens

TLR directly induce innate immune responses by sensing a variety of microbial components and are critical for the fine‐tuning of subsequent adaptive immune responses. However, their impact and mechanism of action on antibody responses against bacterial antigens are not yet fully understood. Salmonella enterica serovar Typhi (S. typhi) porins have been characterized as inducers of long‐lasting specific antibody responses in mice. In this report, we show that immunization of TLR4‐deficient (TLR4−/−), myeloid differentiating gene 88‐deficient and Toll/IL‐R domain‐containing adaptor‐inducing IFN‐β‐deficient mice with S. typhi porins led to significantly reduced B‐cell responses. TLR2−/− mice, as well, showed reduced IgG titers with a more pronounced impairment in the production of IgG3 anti‐porins antibodies. Adoptive transfer of TLR2−/−‐ or TLR4−/−‐B cells into B‐cell‐deficient mice revealed a direct effect of TLR4 on B cells for the primary IgM response, whereas stimulation of B cells via TLR2 was important for IgG production. Furthermore, S. typhi porins were found to efficiently elicit maturation of CD11c+ conventional DC. Taken together, S. typhi porins represent not only a suitable B‐cell antigen for vaccination, but exhibit potent TLR‐dependent stimulatory functions on B cells and DC, which help to further enhance and shape the antibody response.

Translating innate response into long-lasting antibody response by the intrinsic antigen-adjuvant properties of papaya mosaic virus

Identifying the properties of a molecule involved in the efficient activation of the innate and adaptive immune responses that lead to long‐lasting immunity is crucial for vaccine and adjuvant development. Here we show that the papaya mosaic virus (PapMV) is recognized by the immune system as a pathogen‐associated molecular pattern (PAMP) and as an antigen in mice (Pamptigen). A single immunization of PapMV without added adjuvant efficiently induced both cellular and specific long‐lasting antibody responses. PapMV also efficiently activated innate immune responses, as shown by the induction of lipid raft aggregation, secretion of pro‐inflammatory cytokines, up‐regulation of co‐stimulatory molecules on dendritic cells and macrophages, and long‐lasting adjuvant effects upon the specific antibody responses to model antigens. PapMV mixed with Salmonella enterica serovar Typhi (S. typhi) outer membrane protein C increased its protective capacity against challenge with S. typhi, revealing the intrinsic adjuvant properties of PapMV in the induction of immunity. Antigen‐presenting cells loaded with PapMV efficiently induced antibody responses in vivo, which may link the innate and adaptive responses observed. PapMV recognition as a Pamptigen might be translated into long‐lasting antibody responses and protection observed. These properties could be used in the development of new vaccine platforms.

Heat shock protein 70 down-regulates the production of toll-like receptor-induced pro-inflammatory cytokines by a heat shock factor-1/constitutive heat shock element-binding factor-dependent mechanism

BackgroundHeat shock protein 70 (Hsp70) is an intracellular chaperone protein with regulatory and cytoprotective functions. Hsp70 can also be found in the extracellular milieu, as a result of active secretion or passive release from damaged cells. The role of extracellular Hsp70 is not fully understood. Some studies report that it activates monocytes, macrophages and dendritic cells through innate immune receptors (such as Toll-like receptors, TLRs), while others report that Hsp70 is a negative regulator of the inflammatory response. In order to address this apparent inconsistency, in this study we evaluated the response of human monocytes to a highly purified recombinant Hsp70.MethodsHuman peripheral blood monocytes were stimulated with Hsp70, alone or in combination with TLR agonists. Cytokines were quantified in culture supernatants, their mRNAs were measured by RT-PCR, and the binding of transcription factors was evaluated by electrophoretic mobility shift assay (EMSA). Kruskal-Wallis test or one-way or two-way ANOVA were used to analyze the data.ResultsThe addition of Hsp70 to TLR-activated monocytes down-regulated TNF-α as well as IL-6 levels. This effect was independent of a physical interaction between Hsp70 and TLR agonists; instead it resulted of changes at the TNF-α gene expression level. The decrease in TNF-α expression correlated with the binding of HSF-1 (heat shock transcription factor 1, a transcription factor activated in response to Hsp70) and CHBF (constitutive HSE-binding factor) to the TNF-α gene promoter.ConclusionExtracellular Hsp70 negatively regulates the production of pro-inflammatory cytokines of monocytes exposed to TLR agonists and contributes to dampen the inflammatory response.

IFN-γ–Producing CD4+ T Cells Promote Generation of Protective Germinal Center–Derived IgM+ B Cell Memory against Salmonella Typhi

Abs play a significant role in protection against the intracellular bacterium Salmonella Typhi. In this article, we investigated how long-term protective IgM responses can be elicited by a S. Typhi outer-membrane protein C– and F–based subunit vaccine (porins). We found that repeated Ag exposure promoted a CD4+ T cell–dependent germinal center reaction that generated mutated IgM-producing B cells and was accompanied by a strong expansion of IFN-γ–secreting T follicular helper cells. Genetic ablation of individual cytokine receptors revealed that both IFN-γ and IL-17 are required for optimal germinal center reactions and production of porin-specific memory IgM+ B cells. However, more profound reduction of porin-specific IgM B cell responses in the absence of IFN-γR signaling indicated that this cytokine plays a dominant role. Importantly, mutated IgM mAbs against porins exhibited bactericidal capacity and efficiently augmented S. Typhi clearance. In conclusion, repeated vaccination with S. Typhi porins programs type I T follicular helper cell responses that contribute to the diversification of B cell memory and promote the generation of protective IgM Abs.

Salmonella Typhi OmpS1 and OmpS2 porins are potent protective immunogens with adjuvant properties

Salmonella enterica serovar Typhi (S. Typhi) is the causal agent of typhoid fever, a disease that primarily affects developing countries. Various antigens from this bacterium have been reported to be targets of the immune response. Recently, the S. Typhi genome has been shown to encode two porins – OmpS1 and OmpS2 – which are expressed at low levels under in vitro culture conditions. In this study, we demonstrate that immunizing mice with either OmpS1 or OmpS2 induced production of specific, long‐term antibody titres and conferred protection against S. Typhi challenge; in particular, OmpS1 was more immunogenic and conferred greater protective effects than OmpS2. We also found that OmpS1 is a Toll‐like receptor 4 (TLR4) agonist, whereas OmpS2 is a TLR2 and TLR4 agonist. Both porins induced the production of tumour necrosis factor and interleukin‐6, and OmpS2 was also able to induce interleukin‐10 production. Furthermore, OmpS1 induced the over‐expression of MHC II molecules in dendritic cells and OmpS2 induced the over‐expression of CD40 molecules in macrophages and dendritic cells. Co‐immunization of OmpS1 or OmpS2 with ovalbumin (OVA) increased anti‐OVA antibody titres, the duration and isotype diversity of the OVA‐specific antibody response, and the proliferation of T lymphocytes. These porins also had adjuvant effects on the antibody response when co‐immunized with either the Vi capsular antigen from S. Typhi or inactivated 2009 pandemic influenza A(H1N1) virus [A(H1N1)pdm09]. Taken together, the data indicate that OmpS1 and OmpS2, despite being expressed at low levels under in vitro culture conditions, are potent protective immunogens with intrinsic adjuvant properties.

Antibody Persistence in Adults Two Years after Vaccination with an H1N1 2009 Pandemic Influenza Virus-Like Particle Vaccine.

The influenza virus is a human pathogen that causes epidemics every year, as well as potential pandemic outbreaks, as occurred in 2009. Vaccination has proven to be sufficient in the prevention and containment of viral spreading. In addition to the current egg-based vaccines, new and promising vaccine platforms, such as cell culture-derived vaccines that include virus-like particles (VLPs), have been developed. VLPs have been shown to be both safe and immunogenic against influenza infections. Although antibody persistence has been studied in traditional egg-based influenza vaccines, studies on antibody response durations induced by VLP influenza vaccines in humans are scarce. Here, we show that subjects vaccinated with an insect cell-derived VLP vaccine, in the midst of the 2009 H1N1 influenza pandemic outbreak in Mexico City, showed antibody persistence up to 24 months post-vaccination. Additionally, we found that subjects that reported being revaccinated with a subsequent inactivated influenza virus vaccine showed higher antibody titres to the pandemic influenza virus than those who were not revaccinated. These findings provide insights into the duration of the antibody responses elicited by an insect cell-derived pandemic influenza VLP vaccine and the possible effects of subsequent influenza vaccination on antibody persistence induced by this VLP vaccine in humans.

Subversion of innate and adaptive immune activation induced by structurally modified lipopolysaccharide from Salmonella typhimurium

Salmonella are successful pathogens that infect millions of people every year. During infection, Salmonella typhimurium changes the structure of its lipopolysaccharide (LPS) in response to the host environment, rendering bacteria resistant to cationic peptide lysis in vitro. However, the role of these structural changes in LPS as in vivo virulence factors and their effects on immune responses and the generation of immunity are largely unknown. We report that modified LPS are less efficient than wild‐type LPS at inducing pro‐inflammatory responses. The impact of this LPS‐mediated subversion of innate immune responses was demonstrated by increased mortality in mice infected with a non‐lethal dose of an attenuated S. typhimurium strain mixed with the modified LPS moieties. Up‐regulation of co‐stimulatory molecules on antigen‐presenting cells and CD4+ T‐cell activation were affected by these modified LPS. Strains of S. typhimurium carrying structurally modified LPS are markedly less efficient at inducing specific antibody responses. Immunization with modified LPS moiety preparations combined with experimental antigens, induced an impaired Toll‐like receptor 4‐mediated adjuvant effect. Strains of S. typhimurium carrying structurally modified LPS are markedly less efficient at inducing immunity against challenge with virulent S. typhimurium. Hence, changes in S. typhimurium LPS structure impact not only on innate immune responses but also on both humoral and cellular adaptive immune responses.

PD-L1 Expression Induced by the 2009 Pandemic Influenza A(H1N1) Virus Impairs the Human T Cell Response

PD-L1 expression plays a critical role in the impairment of T cell responses during chronic infections; however, the expression of PD-L1 on T cells during acute viral infections, particularly during the pandemic influenza virus (A(H1N1)pdm09), and its effects on the T cell response have not been widely explored. We found that A(H1N1)pdm09 virus induced PD-L1 expression on human dendritic cells (DCs) and T cells, as well as PD-1 expression on T cells. PD-L1 expression impaired the T cell response against A(H1N1)pdm09 by promoting CD8+ T cell death and reducing cytokine production. Furthermore, we found increased PD-L1 expression on DCs and T cells from influenza-infected patients from the first and second 2009 pandemic waves in Mexico City. PD-L1 expression on CD8+ T cells correlated inversely with T cell proportions in patients infected with A(H1N1)pdm09. Therefore, PD-L1 expression on DCs and T cells could be associated with an impaired T cell response during acute infection with A(H1N1)pdm09 virus.

Salmonella Typhi Porins OmpC and OmpF Are Potent Adjuvants for T-Dependent and T-Independent Antigens

Several microbial components, such as bacterial DNA and flagellin, have been used as experimental vaccine adjuvants because of their inherent capacity to efficiently activate innate immune responses. Likewise, our previous work has shown that the major Salmonella Typhi (S. Typhi) outer membrane proteins OmpC and OmpF (porins) are highly immunogenic protective antigens that efficiently stimulate innate and adaptive immune responses in the absence of exogenous adjuvants. Moreover, S. Typhi porins induce the expression of costimulatory molecules on antigen-presenting cells through toll-like receptor canonical signaling pathways. However, the potential of major S. Typhi porins to be used as vaccine adjuvants remains unknown. Here, we evaluated the adjuvant properties of S. Typhi porins against a range of experimental and clinically relevant antigens. Co-immunization of S. Typhi porins with ovalbumin (OVA), an otherwise poorly immunogenic antigen, enhanced anti-OVA IgG titers, antibody class switching, and affinity maturation. This adjuvant effect was dependent on CD4+ T-cell cooperation and was associated with an increase in IFN-γ, IL-17A, and IL-2 production by OVA-specific CD4+ T cells. Furthermore, co-immunization of S. Typhi porins with an inactivated H1N1 2009 pandemic influenza virus experimental vaccine elicited higher hemagglutinating anti-influenza IgG titers, antibody class switching, and affinity maturation. Unexpectedly, co-administration of S. Typhi porins with purified, unconjugated Vi capsular polysaccharide vaccine (Vi CPS)—a T-independent antigen—induced higher IgG antibody titers and class switching. Together, our results suggest that S. Typhi porins OmpC and OmpF are versatile vaccine adjuvants, which could be used to enhance T-cell immune responses toward a Th1/Th17 profile, while improving antibody responses to otherwise poorly immunogenic T-dependent and T-independent antigens.