Description: Certain major histocompatibility complex class I (MHC-I) alleles (e.g., HLA-B*27 ) are enriched among human immunodeficiency virus type 1 (HIV-1)-infected individuals who suppress viremia without treatment (termed "elite controllers" [ECs]). Likewise, Mamu-B*08 expression also predisposes rhesus macaques to control simian immunodeficiency virus (SIV) replication. Given the similarities between Mamu-B*08 and HLA-B*27, SIV-infected Mamu-B*08 + animals provide a model to investigate HLA-B*27-mediated elite control. We have recently shown that vaccination with three immunodominant Mamu-B*08-restricted epitopes (Vif RL8, Vif RL9, and Nef RL10) increased the incidence of elite control in Mamu-B*08 + macaques after challenge with the pathogenic SIVmac239 clone. Furthermore, a correlate analysis revealed that CD8 + T cells targeting Nef RL10 was correlated with improved outcome. Interestingly, this epitope is conserved between SIV and HIV-1 and exhibits a delayed and atypical escape pattern. These features led us to postulate that a monotypic vaccine-induced Nef RL10-specific CD8 + T-cell response would facilitate the development of elite control in Mamu-B*08 + animals following repeated intrarectal challenges with SIVmac239. To test this, we vaccinated Mamu-B*08 + animals with nef inserts in which Nef RL10 was either left intact (group 1) or disrupted by mutations (group 2). Although monkeys in both groups mounted Nef-specific cellular responses, only those in group 1 developed Nef RL10-specific CD8 + T cells. These vaccine-induced effector memory CD8 + T cells did not prevent infection. Escape variants emerged rapidly in the group 1 vaccinees, and ultimately, the numbers of ECs were similar in groups 1 and 2. High-frequency vaccine-induced CD8 + T cells focused on a single conserved epitope and therefore did not prevent infection or increase the incidence of elite control in Mamu-B*08 + macaques. IMPORTANCE Since elite control of chronic-phase viremia is a classic example of an effective immune response against HIV/SIV, elucidating the basis of this phenomenon may provide useful insights into how to elicit such responses by vaccination. We have previously established that vaccine-induced CD8 + T-cell responses against three immunodominant epitopes can increase the incidence of elite control in SIV-infected Mamu-B*08 + rhesus macaques—a model of HLA-B*27-mediated elite control. Here, we investigated whether a monotypic vaccine-induced CD8 + T-cell response targeting the conserved "late-escaping" Nef RL10 epitope can increase the incidence of elite control in Mamu-B*08 + monkeys. Surprisingly, vaccine-induced Nef RL10-specific CD8 + T cells selected for variants within days after infection and, ultimately, did not facilitate the development of elite control. Elite control is, therefore, likely to involve CD8 + T-cell responses against more than one epitope. Together, these results underscore the complexity and multidimensional nature of virologic control of lentivirus infection.

Description: Simian immunodeficiency virus (SIV)-specific CD8 + T cells kill SIV-infected CD4 + T cells in an major histocompatibility complex class I (MHC-I)-dependent manner. However, they are reportedly less efficient at killing SIV-infected macrophages. Since the viral accessory protein Nef has been shown to downregulate MHC-I molecules and enhance cytotoxic T lymphocyte (CTL) evasion in human immunodeficiency virus type 1 (HIV-1)-infected CD4 + T cells, we examined whether Nef played a role in protecting SIV-infected macrophages from killing by SIV-specific CD8 + T cells. To explore the role of Nef in CD8 + T cell evasion, we compared the ability of freshly sorted SIV-specific CD8 + T cells to readily suppress viral replication or eliminate CD4 + T cells or monocyte-derived macrophages infected with SIV variants containing wild-type (WT) or mutated nef genes. As expected, SIV-specific CD8 + T cells suppressed viral replication and eliminated the majority of SIV-infected CD4 + T cells, and this killing was enhanced in CD4 + T cells infected with the nef variants. However, macrophages infected with nef variants that disrupt MHC-I downregulation did not promote rapid killing by freshly isolated CD8 + T cells. These results suggest that mechanisms other than Nef-mediated MHC-I downregulation govern the resistance of SIV-infected macrophages to CD8 + T cell-mediated killing. This study has implications for viral persistence and suggests that macrophages may afford primate lentiviruses some degree of protection from immune surveillance. IMPORTANCE Myeloid cells are permissive for HIV/SIV replication in vitro and may contribute to viral persistence in vivo . While many studies have been geared to understanding how CD8 + T cells control viral replication in CD4 + T cells, the role of these cells in controlling viral replication in macrophages is less clear. Primary, unstimulated CD8 + T cells insignificantly suppress viral replication or eliminate SIV-infected macrophages. Since the viral Nef protein downregulates MHC-I and provides infected cells some degree of protection from CD8 + T cell-mediated effector functions, we evaluated whether Nef may be contributing to the resistance of macrophages to CD8 + T cell suppression. Our results suggest that Nef is not involved in protecting infected macrophages from CD8 + T cell killing and suggest that other mechanisms are involved in macrophage evasion from CD8 surveillance.

Description: Exposure to dengue virus (DENV) is thought to elicit lifelong immunity, mediated by DENV-neutralizing antibodies (nAbs). However, Abs generated by primary infections confer serotype-specific protection, and immunity against other serotypes develops only after subsequent infections. Accordingly, the induction of these nAb responses acquired after serial DENV infections has been a long-sought-after goal for vaccination. Nonetheless, it is still unclear if tetravalent vaccines can elicit or recall nAbs. In this study, we have characterized the responses from a volunteer who had been previously exposed to DENV and was immunized with the live attenuated tetravalent vaccine Butantan-DV, developed by the NIH and Butantan Institute. Eleven days after vaccination, we observed an ~70-fold expansion of the plasmablast population. We generated 21 monoclonal Abs (MAbs) from singly sorted plasmablasts. These MAbs were the result of clonal expansions and had significant levels of somatic hypermutation (SHM). Nineteen MAbs (90.5%) neutralized at least one DENV serotype at concentrations of 1 μg/ml or less; 6 of the 21 MAbs neutralized three or more serotypes. Despite the tetravalent composition of the vaccine, we observed a neutralization bias in the induced repertoire: DENV3 was targeted by 18 of the 19 neutralizing MAbs (nMAbs). Furthermore, the P3D05 nMAb neutralized DENV3 with extraordinary potency (concentration to achieve half-maximal neutralization [Neut 50 ] = 0.03 μg/ml). Thus, the Butantan-DV vaccine engendered a mature, antigen-selected B cell repertoire. Our results suggest that preexisting responses elicited by a previous DENV3 infection were recalled by immunization. IMPORTANCE The dengue epidemic presents a global public health challenge that causes widespread economic burden and remains largely unchecked by existing control strategies. Successful control of the dengue epidemic will require effective prophylactic and therapeutic interventions. Several vaccine clinical efficacy trials are approaching completion, and the chances that one or more live attenuated tetravalent vaccines (LATVs) will be introduced worldwide is higher than ever. While it is widely accepted that dengue virus (DENV)-neutralizing antibody (nAb) titers are associated with protection, the Ab repertoire induced by LATVs remain uncharacterized. Here, we describe the isolation of potent (Neut 50 〈 0.1 μg/ml) nAbs from a DENV-seropositive volunteer immunized with the tetravalent vaccine Butantan-DV, which is currently in phase III trials.

Description: Salmonella enterica serovar Typhimurium DT104 is a recognized food-borne pathogen that displays a multidrug-resistant phenotype and that is associated with systemic infections. At one extreme of the food chain, this bacterium can infect humans, limiting the treatment options available and thereby contributing to increased morbidity and mortality. Although the antibiotic resistance profile is well defined, little is known about other phenotypes that may be expressed by this pathogen at key points across the pork production food chain. In this study, 172 Salmonella enterica serovar Typhimurium DT104/DT104b isolated from an extensive "farm-to-fork" surveillance study, focusing on the pork food chain, were characterized in detail. Isolates were cultured from environmental, processing, retail, and clinical sources, and the study focused on phenotypes that may have contributed to persistence/survival in these different niches. Molecular subtypes, along with antibiotic resistance profiles, tolerance to biocides, motility, and biofilm formation, were determined. As a basis for human infection, acid survival and the ability to utilize a range of energy sources and to adhere to and/or invade Caco-2 cells were also studied. Comparative alterations to biocide tolerance were observed in isolates from retail. l -Tartaric acid and d -mannose-1-phosphate induced the formation of biofilms in a preselected subset of strains, independent of their origin. All clinical isolates were motile and demonstrated an enhanced ability to survive in acidic conditions. Our data report on a diverse phenotype, expressed by S . Typhimurium isolates cultured from the pork production food chain. Extending our understanding of the means by which this pathogen adapts to environmental niches along the "farm-to-fork" continuum will facilitate the protection of vulnerable consumers through targeted improvements in food safety measures.

Description: Nitric oxide (NO) is a proposed component of malaria pathogenesis, and the inducible nitric oxide synthase gene ( NOS2 ) has been associated to malaria susceptibility. We analyzed the role of NOS2 polymorphisms on NO bioavailability and on susceptibility to infection, Plasmodium carrier status and clinical malaria. Two distinct West African sample collections were studied: a population-based collection of 1,168 apparently healthy individuals from the Príncipe Island and a hospital-based cohort of 269 Angolan children. We found that two NOS2 promoter single-nucleotide polymorphism (SNP) alleles associated to low NO plasma levels in noninfected individuals were also associated to reduced risk of pre-erythrocytic infection as measured anti-CSP antibody levels (6.25E–04 〈 P 〈 7.57E–04). In contrast, three SNP alleles within the NOS2 cistronic region conferring increased NO plasma levels in asymptomatic carriers were strongly associated to risk of parasite carriage (8.00E–05 〈 P 〈 7.90E–04). Notwithstanding, three SNP alleles in this region protected from cerebral malaria (7.90E–4 〈 P 〈 4.33E–02). Cohesively, the results revealed a dual regimen in the genetic control of NO bioavailability afforded by NOS2 depending on the infection status. NOS2 promoter variants operate in noninfected individuals to decrease both NO bioavailability and susceptibility to pre-erythrocytic infection. Conversely, NOS2 cistronic variants (namely, rs6505469) operate in infected individuals to increase NO bioavailability and confer increased susceptibility to unapparent infection but protect from cerebral malaria. These findings corroborate the hypothesis that NO anti-inflammatory properties impact on different steps of malaria pathogenesis, explicitly by favoring infection susceptibility and deterring severe malaria syndromes.

Description: Cronobacter species are opportunistic pathogens commonly found in the environment. Among the seven Cronobacter species, Cronobacter sakazakii sequence type 4 (ST-4) is predominantly associated with recorded cases of infantile meningitis. This study reports on a 26-month powdered infant formula (PIF) surveillance program in four production facilities located in distinct geographic regions. The objective was to identify the ST(s) in PIF production environments and to investigate the phenotypic features that support their survival. Of all 168 Cronobacter isolates, 133 were recovered from a PIF production environment, 31 were of clinical origin, and 4 were laboratory type strains. Sequence type 1 ( n = 84 isolates; 63.9%) was the dominant type in PIF production environments. The majority of these isolates clustered with an indistinguishable pulsotype and persisted for at least an 18-month period. Moreover, DNA microarray results identified two phylogenetic lineages among ST-4 strains tested. Thereafter, the ST-1 and -4 isolates were phenotypically compared. Differences were noted based on the phenotypes expressed by these isolates. The ST-1 PIF isolates produced stronger biofilms at both 28°C and 37°C, while the ST-4 clinical isolates exhibited greater swimming activity and increased binding to Congo red dye. Given the fact that PIF is a low-moisture environment and that the clinical environment provides for an interaction between the pathogen and its host, these differences may be consistent with a form of pathoadaptation. These findings help to extend our current understanding of the epidemiology and ecology of Cronobacter species in PIF production environments.

Description: Epidemiological cutoff values (ECVs) for the Cryptococcus neoformans-Cryptococcus gattii species complex versus fluconazole, itraconazole, posaconazole, and voriconazole are not available. We established ECVs for these species and agents based on wild-type (WT) MIC distributions. A total of 2,985 to 5,733 CLSI MICs for C. neoformans (including isolates of molecular type VNI [MICs for 759 to 1,137 isolates] and VNII, VNIII, and VNIV [MICs for 24 to 57 isolates]) and 705 to 975 MICs for C. gattii (including 42 to 260 for VGI, VGII, VGIII, and VGIV isolates) were gathered in 15 to 24 laboratories (Europe, United States, Argentina, Australia, Brazil, Canada, Cuba, India, Mexico, and South Africa) and were aggregated for analysis. Additionally, 220 to 359 MICs measured using CLSI yeast nitrogen base (YNB) medium instead of CLSI RPMI medium for C. neoformans were evaluated. CLSI RPMI medium ECVs for distributions originating from at least three laboratories, which included ≥95% of the modeled WT population, were as follows: fluconazole, 8 μg/ml (VNI, C. gattii nontyped, VGI, VGIIa, and VGIII), 16 μg/ml ( C. neoformans nontyped, VNIII, and VGIV), and 32 μg/ml (VGII); itraconazole, 0.25 μg/ml (VNI), 0.5 μg/ml ( C. neoformans and C. gattii nontyped and VGI to VGIII), and 1 μg/ml (VGIV); posaconazole, 0.25 μg/ml ( C. neoformans nontyped and VNI) and 0.5 μg/ml ( C. gattii nontyped and VGI); and voriconazole, 0.12 μg/ml (VNIV), 0.25 μg/ml ( C. neoformans and C. gattii nontyped, VNI, VNIII, VGII, and VGIIa,), and 0.5 μg/ml (VGI). The number of laboratories contributing data for other molecular types was too low to ascertain that the differences were due to factors other than assay variation. In the absence of clinical breakpoints, our ECVs may aid in the detection of isolates with acquired resistance mechanisms and should be listed in the revised CLSI M27-A3 and CLSI M27-S3 documents.

Description: Clinical breakpoints (CBPs) are not available for the Cryptococcus neoformans-Cryptococcus gattii species complex. MIC distributions were constructed for the wild type (WT) to establish epidemiologic cutoff values (ECVs) for C. neoformans and C. gattii versus amphotericin B and flucytosine. A total of 3,590 amphotericin B and 3,045 flucytosine CLSI MICs for C. neoformans (including 1,002 VNI isolates and 8 to 39 VNII, VNIII, and VNIV isolates) and 985 and 853 MICs for C. gattii , respectively (including 42 to 259 VGI, VGII, VGIII, and VGIV isolates), were gathered in 9 to 16 (amphotericin B) and 8 to 13 (flucytosine) laboratories (Europe, United States, Australia, Brazil, Canada, India, and South Africa) and aggregated for the analyses. Additionally, 442 amphotericin B and 313 flucytosine MICs measured by using CLSI-YNB medium instead of CLSI-RPMI medium and 237 Etest amphotericin B MICs for C. neoformans were evaluated. CLSI-RPMI ECVs for distributions originating in ≥3 laboratories (with the percentages of isolates for which MICs were less than or equal to ECVs given in parentheses) were as follows: for amphotericin B, 0.5 μg/ml for C. neoformans VNI (97.2%) and C. gattii VGI and VGIIa (99.2 and 97.5%, respectively) and 1 μg/ml for C. neoformans (98.5%) and C. gattii nontyped (100%) and VGII (99.2%) isolates; for flucytosine, 4 μg/ml for C. gattii nontyped (96.4%) and VGI (95.7%) isolates, 8 μg/ml for VNI (96.6%) isolates, and 16 μg/ml for C. neoformans nontyped (98.6%) and C. gattii VGII (97.1%) isolates. Other molecular types had apparent variations in MIC distributions, but the number of laboratories contributing data was too low to allow us to ascertain that the differences were due to factors other than assay variation. ECVs may aid in the detection of isolates with acquired resistance mechanisms.

Description: Broadly targeted cellular immune responses are thought to be important for controlling replication of human and simian immunodeficiency viruses (HIV and SIV). However, eliciting such responses by vaccination is complicated by immunodominance, the preferential targeting of only a few of the many possible epitopes of a given antigen. This phenomenon may be due to the coexpression of dominant and subdominant epitopes by the same antigen-presenting cell and may be overcome by distributing these sequences among several different vaccine constructs. Accordingly, we tested whether vaccinating rhesus macaques with "minigenes" encoding fragments of Gag, Vif, and Nef resulted in broadened cellular responses capable of controlling SIV replication. We delivered these minigenes through combinations of recombinant Mycobacterium bovis BCG (rBCG), electroporated recombinant DNA (rDNA) along with an interleukin-12 (IL-12)-expressing plasmid (EP rDNA plus pIL-12), yellow fever vaccine virus 17D (rYF17D), and recombinant adenovirus serotype 5 (rAd5). Although priming with EP rDNA plus pIL-12 increased the breadth of vaccine-induced T-cell responses, this effect was likely due to the improved antigen delivery afforded by electroporation rather than modulation of immunodominance. Indeed, Mamu-A*01 + vaccinees mounted CD8 + T cells directed against only one subdominant epitope, regardless of the vaccination regimen. After challenge with SIVmac239, vaccine efficacy was limited to a modest reduction in set point in some of the groups and did not correlate with standard T-cell measurements. These findings suggest that broad T-cell responses elicited by conventional vectors may not be sufficient to substantially contain AIDS virus replication. IMPORTANCE Immunodominance poses a major obstacle to the generation of broadly targeted, HIV-specific cellular responses by vaccination. Here we attempted to circumvent this phenomenon and thereby broaden the repertoire of SIV-specific cellular responses by vaccinating rhesus macaques with minigenes encoding fragments of Gag, Vif, and Nef. In contrast to previous mouse studies, this strategy appeared to minimally affect monkey CD8 + T-cell immundominance hierarchies, as seen by the detection of only one subdominant epitope in Mamu-A*01 + vaccinees. This finding underscores the difficulty of inducing subdominant CD8 + T cells by vaccination and demonstrates that strategies other than gene fragmentation may be required to significantly alter immunodominance in primates. Although some of the regimens tested here were extremely immunogenic, vaccine efficacy was limited to a modest reduction in set point viremia after challenge with SIVmac239. No correlates of protection were identified. These results reinforce the notion that vaccine immunogenicity does not predict control of AIDS virus replication.

Description: The intracellular protozoan parasite Trypanosoma cruzi is the etiologic agent of Chagas disease, a serious disorder that affects millions of people in Latin America. Cell invasion by T. cruzi and its intracellular replication are essential to the parasite's life cycle and for the development of Chagas disease. Here, we present evidence suggesting the involvement of the host's cyclooxygenase (COX) enzymes during T. cruzi invasion. Pharmacological antagonists for COX-1 (aspirin) and COX-2 (celecoxib) caused marked inhibition of T. cruzi infection when rat cardiac cells were pretreated with these nonsteroidal anti-inflammatory drugs (NSAIDs) for 60 min at 37°C before inoculation. This inhibition was associated with an increase in the production of NO and interleukin-1β and decreased production of transforming growth factor β (TGF-β) by cells. Taken together, these results indicate that COX-1 more than COX-2 is involved in the regulation of anti- T. cruzi activity in cardiac cells, and they provide a better understanding of the influence of TGF-β-interfering therapies on the innate inflammatory response to T. cruzi infection and may represent a very pertinent target for new therapeutic treatments of Chagas disease.