Seasonal H3N2 influenza A virus fails to enhance Staphylococcus aureus co-infection in a non-human primate respiratory tract infection model.

Staphylococcus aureus community-acquired pneumonia is often associated with influenza or an influenza-like syndrome. Morbidity and mortality due to methicillin-resistant S. aureus (MRSA) or influenza and pneumonia, which includes bacterial co-infection, are among the top causes of death by infectious diseases in the United States. We developed a non-lethal influenza A virus (IAV) (H3N2)/S. aureus co-infection model in cynomolgus macaques (Macaca fascicularis) to test the hypothesis that seasonal IAV infection predisposes non-human primates to severe S. aureus pneumonia. Infection and disease progression were monitored by clinical assessment of animal health; analysis of blood chemistry, nasal swabs, and X-rays; and gross pathology and histopathology of lungs from infected animals. Seasonal IAV infection in healthy cynomolgus macaques caused mild pneumonia, but unexpectedly, did not predispose these animals to subsequent severe infection with the community-associated MRSA clone USA300. We conclude that in our co-infection model, seasonal IAV infection alone is not sufficient to promote severe S. aureus pneumonia in otherwise healthy non-human primates. The implication of these findings is that comorbidity factors in addition to IAV infection are required to predispose individuals to secondary S. aureus pneumonia.

Interactome analysis of longitudinal pharyngeal infection of cynomolgus macaques by group A Streptococcus.

Relatively little is understood about the dynamics of global host-pathogen transcriptome changes that occur during bacterial infection of mucosal surfaces. To test the hypothesis that group A Streptococcus (GAS) infection of the oropharynx provokes a distinct host transcriptome response, we performed genome-wide transcriptome analysis using a nonhuman primate model of experimental pharyngitis. We also identified host and pathogen biological processes and individual host and pathogen gene pairs with correlated patterns of expression, suggesting interaction. For this study, 509 host genes and seven biological pathways were differentially expressed throughout the entire 32-day infection cycle. GAS infection produced an initial widespread significant decrease in expression of many host genes, including those involved in cytokine production, vesicle formation, metabolism, and signal transduction. This repression lasted until day 4, at which time a large increase in expression of host genes was observed, including those involved in protein translation, antigen presentation, and GTP-mediated signaling. The interactome analysis identified 73 host and pathogen gene pairs with correlated expression levels. We discovered significant correlations between transcripts of GAS genes involved in hyaluronic capsule production and host endocytic vesicle formation, GAS GTPases and host fibrinolytic genes, and GAS response to interaction with neutrophils. We also identified a strong signal, suggesting interaction between host gammadelta T cells and genes in the GAS mevalonic acid synthesis pathway responsible for production of isopentenyl-pyrophosphate, a short-chain phospholipid that stimulates these T cells. Taken together, our results are unique in providing a comprehensive understanding of the host-pathogen interactome during mucosal infection by a bacterial pathogen.

Longitudinal analysis of the group A Streptococcus transcriptome in experimental pharyngitis in cynomolgus macaques.

Identification of the genetic events that contribute to host-pathogen interactions is important for understanding the natural history of infectious diseases and developing therapeutics. Transcriptome studies conducted on pathogens have been central to this goal in recent years. However, most of these investigations have focused on specific end points or disease phases, rather than analysis of the entire time course of infection. To gain a more complete understanding of how bacterial gene expression changes over time in a primate host, the transcriptome of group A Streptococcus (GAS) was analyzed during an 86-day infection protocol in 20 cynomolgus macaques with experimental pharyngitis. The study used 260 custom Affymetrix (Santa Clara, CA) chips, and data were confirmed by TaqMan analysis. Colonization, acute, and asymptomatic phases of disease were identified. Successful colonization and severe inflammation were significantly correlated with an early onset of superantigen gene expression. The differential expression of two-component regulators covR and spy0680 (M1_spy0874) was significantly associated with GAS colony-forming units, inflammation, and phases of disease. Prophage virulence gene expression and prophage induction occurred predominantly during high pathogen cell densities and acute inflammation. We discovered that temporal changes in the GAS transcriptome were integrally linked to the phase of clinical disease and host-defense response. Knowledge of the gene expression patterns characterizing each phase of pathogen-host interaction provides avenues for targeted investigation of proven and putative virulence factors and genes of unknown function and will assist vaccine research.

Extracellular deoxyribonuclease made by group A Streptococcus assists pathogenesis by enhancing evasion of the innate immune response.

Many pathogenic bacteria produce extracellular DNase, but the benefit of this enzymatic activity is not understood. For example, all strains of the human bacterial pathogen group A Streptococcus (GAS) produce at least one extracellular DNase, and most strains make several distinct enzymes. Despite six decades of study, it is not known whether production of DNase by GAS enhances virulence. To test the hypothesis that extracellular DNase is required for normal progression of GAS infection, we generated seven isogenic mutant strains in which the three chromosomal- and prophage-encoded DNases made by a contemporary serotype M1 GAS strain were inactivated. Compared to the wild-type parental strain, the isogenic triple-mutant strain was significantly less virulent in two mouse models of invasive infection. The triple-mutant strain was cleared from the skin injection site significantly faster than the wild-type strain. Preferential clearance of the mutant strain was related to the differential extracellular killing of the mutant and wild-type strains, possibly through degradation of neutrophil extracellular traps, innate immune structures composed of chromatin and granule proteins. The triple-mutant strain was also significantly compromised in its ability to cause experimental pharyngeal disease in cynomolgus macaques. Comparative analysis of the seven DNase mutant strains strongly suggested that the prophage-encoded SdaD2 enzyme is the major DNase that contributes to virulence in this clone. We conclude that extracellular DNase activity made by GAS contributes to disease progression, thereby resolving a long-standing question in bacterial pathogenesis research.

Group A Streptococcus gene expression in humans and cynomolgus macaques with acute pharyngitis.

The molecular mechanisms used by group A Streptococcus (GAS) to survive on the host mucosal surface and cause acute pharyngitis are poorly understood. To provide new information about GAS host-pathogen interactions, we used real-time reverse transcription-PCR (RT-PCR) to analyze transcripts of 17 GAS genes in throat swab specimens taken from 18 pediatric patients with pharyngitis. The expression of known and putative virulence genes and regulatory genes (including genes in seven two-component regulatory systems) was studied. Several known and previously uncharacterized GAS virulence gene regulators were highly expressed compared to the constitutively expressed control gene proS. To examine in vivo gene transcription in a controlled setting, three cynomolgus macaques were infected with strain MGAS5005, an organism that is genetically representative of most serotype M1 strains recovered from pharyngitis and invasive disease episodes in North America and Western Europe. These three animals developed clinical signs and symptoms of GAS pharyngitis and seroconverted to several GAS extracellular proteins. Real-time RT-PCR analysis of throat swab material collected at intervals throughout a 12-day infection protocol indicated that expression profiles of a subset of GAS genes accurately reflected the profiles observed in the human pediatric patients. The results of our study demonstrate that analysis of in vivo GAS gene expression is feasible in throat swab specimens obtained from infected human and nonhuman primates. In addition, we conclude that the cynomolgus macaque is a useful nonhuman primate model for the study of molecular events contributing to acute pharyngitis caused by GAS.

Rabies DNA vaccination of non-human primates: post-exposure studies using gene gun methodology that accelerates induction of neutralizing antibody and enhances neutralizing antibody titers.

Pre-exposure DNA vaccination protects non-human primates against rabies virus. Post-exposure protection of monkeys against rabies virus by DNA vaccination has not been attempted. Presumably, post-exposure experiments have not been undertaken because neutralizing antibody is usually slow to be induced after DNA vaccination. In this study, we initially attempted to accelerate the induction of neutralizing antibody by varying the route and site of DNA vaccination and booster frequency. Gene gun (GG) vaccinations above axillary and inguinal lymph nodes or in ear pinnae generated higher levels of neutralizing antibody than intradermal (ID) needle vaccinations in the pinnae. Concurrent GG booster vaccinations above axillary and inguinal lymph nodes and in ear pinnae, 3 days after primary vaccination, accelerated detectable neutralizing antibody. GG booster vaccinations also resulted in higher neutralizing antibody levels and increased the durability of this response. Post-exposure vaccination with DNA or the human diploid cell vaccine (HDCV), in combination with an one-time treatment with human rabies immune globulin (HRIG), protected 50 and 75% of the monkeys, respectively, as compared to 75% mortality of the controls. These data will be useful for the refinement, development, and implementation of future pre- and post-exposure rabies DNA vaccination studies.

Meal-induced increase in plasma gastrin immunoreactivity in the rhesus monkey (Macaca mulatta).

The hormonal response to food intake in rodents, dogs, and humans involves gastrin and cholecystokinin, structurally related peptides present in plasma, gut, and brain. In order to determine the time course of plasma gastrin response in a nonhuman primate, six overnight-fasted adult male rhesus monkeys were offered a meal of bananas (11g) and peanut butter (1 Tbsp) or a fresh water bottle in a crossover design. All animals completely consumed the meal within 10 min. Compared to non-fed control levels, plasma gastrin was significantly elevated (52 ± 11 pM vs. 32 ± 9 pM, means ± S. E. M.) from 10 to 45 min post-ingestion, but returned to near basal fasted level by 120 min. Levels of gastrin in tissue samples (n = 2) were highest in the antrum of the stomach, with decreasing amounts measured in the upper and lower duodenum, respectively. The results demonstrate that the plasma concentration and response to a meal of rhesus monkey gastrin are similar to those of other mammalian species. However, the high concentrations of gastrin found in duodenum are thus far unique to Macaca mulatta and humans.

Placental transfer of synthetic and endogenous estrogen conjugates in the rhesus monkey (Macaca mulatta).

The placental transfer of conjugated and nonconjugated estrogens was compared in the pregnant rhesus monkey. Placement of catheters in the maternal and fetal circulation allowed for the sampling of blood after the administration of radiolabeled naturally occurring and synthetic estrogens to mother or fetus. In all cases, nonconjugated-estrogen placental transfer was greater than conjugated-estrogen transfer. Comparison of the conjugated estrogens suggested that diethylstilbestrol-monoglucuronide (DESG) was transferred less efficiently than the estrone-sulfate (E, S). High-performance liquid chromatog-raphy (HPLC) of selected plasma samples revealed that 50-90% of the E, S observed in the maternal circulation was cleaved after fetal administration. In contrast, HPLC of maternal or fetal plasma samples after DESG administration revealed only intact DESG. These results emphasize differences in the placental transfer of the synthetic and naturally occurring estrogen hormones.