In the case-control study of the RV144 vaccine trial, the levels of antibodies to the V1V2 region of the gp120 envelope glycoprotein were found to correlate inversely with risk of HIV infection. This recent demonstration of the potential role of V1V2 as a vaccine target has catapulted this region into the focus of HIV-1 research. We previously described seven human monoclonal antibodies (mAbs) derived from HIV-infected individuals that are directed against conformational epitopes in the V1V2 domain. In this study, using lysates of SF162 pseudoviruses carrying V1V2 mutations, we mapped the epitopes of these seven mAbs. All tested mAbs demonstrated a similar binding pattern in which three mutations  abrogated binding of at least six of the seven mAbs to ≤15% of SF162 wildtype binding. Binding of six or all of the mAbs was reduced to ≤50% of wildtype by single substitutions at seven positions , while one change, V181I, increased the binding of all mAbs. When mapped onto a model of V2, our results suggest that the epitope of the conformational V2 mAbs is located mostly in the disordered region of the available crystal structure of V1V2, overlapping and surrounding the α4β7 binding site on V2. In 2009, the RV144 clinical vaccine trial provided the first success in the development of an effective HIV vaccine, demonstrating a 31.2% efficacy at preventing HIV-1 infection in vaccine recipients + T cells + T cells to the gut mucosa + T cells, α4β7 has been shown to co-localize with CD4 and CCR5 in the cell membrane in vitro viral entry or replication, α4β7 interacts with gp120 proteins from diverse HIV-1 subtypes, suggesting that this integrin may play a key role in mucosal HIV-1 transmission + T cells by full-length infectious molecular clones of subtype C viruses, suggesting that differences in binding may exist between gp120 proteins and the trimeric envelopes present on virions V2 can bind to α4β7, an integrin that is expressed strongly on activated CD4Antibodies targeting the V2 region are found in approximately 20–45% of HIV-1 infected individuals Case A2-gp70, and the RV144-derived V2 mAbs react with this reagent much more weakly or not at all The conformation-dependent V2 mAbs, namely mAbs 697, 830A, 1357D, 1361, 1393A, 2158, and 2297, display broad immunologic cross-reactivity with subtype A, AG, B, and C recombinant gp120 proteins and weak neutralization of Tier 1 pseudoviruses This study was approved by the New York University School of Medicine Institutional Review Board.Point mutations were introduced in the V1V2 region of plasmid pCAGGS SF162 gp160 (NIH AIDS Reagent Program) Wildtype or mutant pCAGGS SF162 gp160 plasmids were used to co-transfect 293T human embryonic kidney cells  with a Δenv backbone plasmid (pNL4.3Δenv or pSG3Δenv) at optimal backbone to envelope ratios, using Fugene HD  according to the manufacturer's protocol. After 48–72 h, supernatants containing the secreted mutant or wildtype pseudoviruses were harvested, filtered, aliquoted and stored at −80°C. Before use in binding assays, pseudovirus supernatants were lysed by mixing with 0.1 volumes of 10% Triton X-100 in PBS.Binding Assays were carried out as previously described Binding (OD405 readings) of each conformational V2 mAb to each mutant pseudovirus was normalized to the CD4-IgG2 binding (OD405) of the same mutant pseudovirus. This controlled for possible differences in virion numbers among the pseudovirus lysates. Binding of wildtype pseudovirus lysate was included on each plate and set as 100%.The conformational V2 mAbs used in this study  were produced in this laboratory and have been described previously de novo loop modeling protocols A full length V1V2 homology model for SF162 was created by using the template crystal structure of the 1FD6-V1V2-scaffold complexed with Fab PG9 (PDB Code: 3U4E). The SF162 sequence was threaded over the V1V2 template coordinates using Rosetta 3.4 Amino acids were exchanged by introducing point mutations at 13 different locations in the V2 region of the gp120 glycoprotein of SF162, starting at position 160 and continuing through position 193 . The means of 3-5 experiments are shown with the standard errors of the mean. White bars show mutations in C2 and C4 regions of gp120 and were used as controls.(TIF)Click here for additional data file.Table S1Mutations used for V2 mapping of monoclonal antibodies.(DOCX)Click here for additional data file.


Staphylococcus aureus (CA-MRSA) are associated with a marked and prolonged host inflammatory response. In a sepsis simulation model, we tested whether the anesthetic ketamine inhibits the macrophage TNF response to antibiotic-exposed CA-MRSA bacteria via its antagonism of N-methyl-D-aspartate (NMDA) receptors. RAW264.7 cells were stimulated for 18 hrs with 105 to 107 CFU/mL inocula of either of two prototypical CA-MRSA isolates, USA300 strain LAC and USA400 strain MW2, in the presence of either vancomycin or daptomycin. One hour before bacterial stimulation, ketamine was added with or without MK-801 , APV , NMDA, or combinations of these agents. Supernatants were collected and assayed for TNF concentration by ELISA.Infections caused by community-associated strains of methicillin-resistant RAW264.7 cells exposed to either LAC or MW2 in the presence of daptomycin secreted less TNF than in the presence of vancomycin. The addition of ketamine inhibited macrophage TNF secretion after stimulation with either of the CA-MRSA isolates  in the presence of either antibiotic. The NMDA inhibitors, MK-801 and APV, also suppressed macrophage TNF secretion after stimulation with either of the antibiotic-exposed CA-MRSA isolates, and the effect was not additive or synergistic with ketamine. The addition of NMDA substrate augmented TNF secretion in response to the CA-MRSA bacteria, and the addition of APV suppressed the effect of NMDA in a dose-dependent fashion.Ketamine inhibits TNF secretion by MRSA-stimulated RAW264.7 macrophages and the mechanism likely involves NMDA receptor antagonism. These findings may have therapeutic significance in MRSA sepsis. Staphylococcus aureus (CA-MRSA) present a major public health problem because of recent increases in the incidence of these infections [Staphylococcus aureus is now the most important cause of serious and fatal infection in the United States [Infections caused by community-associated strains of methicillin-resistant fections ,2. In a d States . The prod States . In 2003d States ,6.CA-MRSA isolates express many virulence factors ,8, incluS. aureus  in the presence of daptomycin (vs. vancomycin) secreted less TNF and accumulated less inducible nitric oxide synthase (iNOS) protein [Monocytes and macrophages play important roles in host defense against staphylococci and other pyogenic bacteria , but exc protein . Vancomy protein . Daptomy protein ,14. The  protein ,16. Therin vivo and in vitro [in vitro after exposure to staphylococcal enterotoxin B [Staphylococcus aureus (MRSA). Given the important role of TNF in sepsis [Ketamine is one of the recommended anesthetics in pediatric septic shock -19, whicin vitro ,23,25. Totoxin B . The mecotoxin B -25.The pn sepsis -29, and Staphylococcus aureus. Because S. aureus is able to "attack" or form pores in macrophages, TNF secretion occurs even in the absence of TLR 2 and TLR4 sensors  [Although membrane-bound Toll-like receptors (TLR2 and TLR4) are essential for lipopolysaccharide (LPS)-induced TNF production , this iseptides) . TherefoWe also tested the effects of two chemically unrelated NMDA receptor antagonists, the anti-convulsant MK-801 (dizocilpine) ,33, a noFor these studies, we utilized two well-characterized clinical isolates: LAC (Los Angeles County), representative of the USA300 group of organisms and closely related to the dominant CA-MRSA clone associated with soft tissue infections and serious invasive disease in the Memphis area , and MW25-107 CFU/mL) was studied, based upon our previously published data with other CA-MRSA strains [Bacteria were grown to late logarithmic phase at 37°C in tryptic soy broth  and washed three times in endotoxin-free phosphate-buffered saline. Concentrations were determined by colony counts. A range of concentrations of bacteria (10 strains  and our Minimum inhibitory concentrations (MICs) for these strains were determined by the microbiology laboratory at Le Bonheur Children's Hospital using the E-test method: both strains were fully susceptible to vancomycin and daptomycin .6 cells per well.RAW264.7 murine macrophage-like cells were purchased from the ATCC and cultured in Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum  and 2 mM glutamine . Experiments were done in 24-well tissue culture plates  with 1 × 105-107 CFU/mL). Cells were then incubated for 18 hours. Daptomycin was obtained from Cubist Pharmaceuticals . Vancomycin was purchased via the Department of Pharmacy at Le Bonheur Children's Hospital (LBCH) from Hospira . Clinically achievable concentrations of each of the antibiotics, as previously tested in our laboratory [Either vancomycin or daptomycin was added to the cell cultures immediately before the addition of live staphylococci  of optically active isomers (R and L) of this drug, purchased from the LBCH pharmacy. Emphasis in the experiment was placed on correlation with the clinical situation; thus racemic ketamine, the most commonly clinically used product, was selected. Dizocilpine (MK-801), APV and NMDA were purchased from Sigma Chemical Co. .These experiments were repeated in parallel in the presence of ketamine (100 μM) and/or MK-801 , APV  or 3 mM ("high"), or NMDA (30 μM). The modulation of MRSA-stimulated macrophage TNF production by ketamine was subsequently examined also at a range of concentrations of 10 μΜ, 50 μΜ, 100 μΜ and 150 μΜ. The selected concentration (100 μM) is based on the achievable anesthetic concentrations -39 and o2 = 0.84, 0.87 and 0.93, respectively). We focused on TNF secretion for these studies.After incubation, cell-free supernatants were collected and assayed for TNF concentrations by using a solid-phase sandwich enzyme-linked immunosorbent assay as specified by the manufacturer . TNF is a key cytokine produced by macrophages during MRSA stimulation. In our preliminary studies, we also measured secretion of other cytokines and found that IL-1, IL-6, and NO secretion were strongly correlated with TNF secretion in response to these bacteria  were conducted to determine whether the inhibition increased over time. In the multiple comparison experiments (ketamine and MK 801 synergistic action), TNF was measured from at least four well replicates. All experiments were performed separately for LAC and for MW2 MRSA strains. There is an intrinsic experimental variation of absolute values of TNF production (up to 25%) because of cell culture and macrophage growth characteristics.For the single comparison experiments (ketamine or MK 801 or APV), TNF secretion measurements were validated with an average of at least three well replicates and each of the experiments was repeated at least three times . The four preliminary runs and all the exposures  where the inocula were different from 10a priori) contrasts, i.e., we set all our comparisons in advance of multiple setting experiments. Significant differences were presumed at a probability value of p < 0.05. The results were graphed using error bars with 95% confidence intervals. Differences in the means were estimated either with asymptotic techniques for normally distributed data or bootstrapping techniques for non-normally distributed data.The design was composed of factorial multiple measurements and the results were analyzed according to a mixed linear model, (GLIMMIX) SAS 9.2  and R 2.9.1 and ggplot2 software. We set pre-planned , suggesting that this effect of daptomycin is conserved in many different S. aureus isolates.As previously observed with two USA300 CA-MRSA strains isolated from Memphis children with invasive staphylococcal infections , macrophThe addition of ketamine (100 μΜ) to macrophage cell cultures inhibited TNF secretion in response to vancomycin- or daptomycin-exposed CA-MRSA isolates Figure . The effp < 0.05), i.e., from 33,085 ± 867 pg/mL to 23,347 ± 862 pg/mL. Pre-incubation with ketamine led to a similar reduction  in macrophage TNF secretion response after stimulation with vancomycin-exposed LAC .In the initial experiments we analyzed the effect of one hour pre-incubation with ketamine on the macrophage response to vancomycin-exposed CA-MRSA bacteria (MW2 and LAC). In response to vancomycin-exposed MW2, pre-incubation with ketamine reduced macrophage TNF secretion by approximately 29% (p < 0.05) or LAC , also inhibited TNF secretion by these cells after stimulation with antibiotic-exposed CA-MRSA strains . MK-801 inhibited macrophage TNF secretion in response to daptomycin-exposed LAC by approximately 33% .Pre-incubation with MK-801 also significantly inhibited macrophage TNF secretion in response to daptomycin-treated MW2 or LAC  alone. Figure p < 0.05, Figure p < 0.05), and this effect was blocked by ketamine and by the competitive NMDA receptor antagonist, APV  and was greater at concentrations of 50 -150 μM Figure .We also examined the kinetics of inhibition of macrophage TNF secretion by incubating RAW264.7 cells for 6, 10, 14, 18 and 24 hours after exposure to ketamine at a concentration of 100 μM 1 hour prior to stimulation with vancomycin-exposed LAC or MW2. We found that the magnitude of suppression of TNF secretion was similar at all times studied  and LAC (USA300) bacteria and was similar in the presence of either vancomycin (a lytic antibiotic associated with a greater TNF response to the bacteria) or daptomycin (a non-lytic antibiotic associated with a blunted TNF response to the bacteria). Our data suggest that ketamine administration to macrophages stimulated by CA-MRSA is associated with blunting of the TNF response to these virulent pathogens, and suggest that these findings may have therapeutic significance in MRSA sepsis. Furthermore, these data confirm and extend our previous observations that CA-MRSA bacteria exposed to daptomycin (versus vancomycin) trigger less TNF secretion by macrophages. The potentially beneficial anti-inflammatory effects of daptomycin and ketamine were additive  to daptomycin (compared with vancomycin) led to a less pronounced macrophage inflammatory response, characterized by diminished secretion of TNF and reduced accumulation of the inducible nitric oxide synthase (iNOS) [In a previous study, we found that exposure of either of two CA-MRSA strains isolated from Memphis children (or any of four other e (iNOS) . In thisThe mechanism(s) responsible for the anti-inflammatory properties of ketamine are not known, but its neurological and psychotropic actions are believed primarily to be mediated by antagonism of NMDA receptors ,47. GlutWe found that MK-801 and APV also inhibited macrophage TNF secretion in response to antibiotic-treated MW2 or LAC cells. The magnitude of the inhibition by MK-801 (approximately 30%) and APV (25-35%) was comparable to that observed with ketamine (18-34%), and combinations of MK-801 and ketamine or of APV and ketamine did not exhibit additive or synergistic inhibition of TNF secretion. Furthermore, adding NMDA led to augmented macrophage TNF secretion in response to antibiotic-treated CA-MRSA bacteria, and the NMDA receptor antagonist, APV, blocked this effect. The suppression of TNF induced by ketamine was observed across a range of concentrations and throughout the incubation period.Our study has its limitations. To translate the present findings, we are currently working on a clinical model to assess the clinical significance of ketamine's anti-inflammatory effects in patients with bacterial sepsis. Although studies of the effect of ketamine on macrophage responses to purified bacterial components such as Gram-negative lipopolysaccharide (LPS) or Gram-positive lipoteichoic acid (LTA) are instructive ,24,49, wIn the battle of sepsis everything counts. Adjunctive therapies of sepsis are greatly needed. Studies in animal models and clinical trials will be required to determine whether the anti-inflammatory effects of ketamine and/or other agents that block NMDA receptors could be beneficial in the treatment of severe staphylococcal infections.Staphylococcus aureus; NMDA: N-methyl-D-aspartate; HA-MRSA: hospital-associated methicillin-resistant Staphylococcus aureus; iNOS: nitric oxide synthase; LPS: lipopolysaccharide; LTA: lipoteichoic acid; PVL: Panton-Valentine leukocidin; PSM: phenol-soluble modulins; APV: D-2-amino-5-phosphono-valerate; MK-801: dizocilpine; CFU: colony forming units; IL 1-12: interleukin 1-12; Le Bonheur Children's Hospital (LBCH)CA-MRSA: community-associated methicillin-resistant The authors declare that they have no competing interests.TS, RKHS, CAA, EM, LL and BSW contributed to acquisition of data. MSR was the medication consultant. TS, EM and BKE contributed to the conception and design and interpretation of the data. TS contributed to the statistical analysis. TS, EM and BKE gave the final approval.National Society of Critical Care meeting in Nashville, TN, Feb. 2009Southern Society of Clinical Investigation/American Federation for Medical Research meeting in New Orleans, LA, Feb. 2009Pediatric Academic Societies' research meeting in Baltimore, MD, May 2009American Academy of Pediatrics Section of Critical Care, Washington DC, October 2009


Salmonella infection in pigs, little is known about the associated dynamics of fecal shedding and immunity. In this study, we investigated the transitions of pigs through the states of Salmonella fecal shedding and immune response post-Salmonella inoculation as affected by the challenge dose and serotype. Continuous-time multistate Markov models were developed using published experimental data. The model for shedding had four transient states, of which two were shedding (continuous and intermittent shedding) and two non-shedding (latency and intermittent non-shedding), and one absorbing state representing permanent cessation of shedding. The immune response model had two transient states representing responses below and above the seroconversion level. The effects of two doses [low (0.65×106 CFU/pig) and high (0.65×109 CFU/pig)] and four serotypes  on the models' transition intensities were evaluated using a proportional intensities model. Results indicated statistically significant effects of the challenge dose and serotype on the dynamics of shedding and immune response. The time spent in the specific states was also estimated. Continuous shedding was on average 10–26 days longer, while intermittent non-shedding was 2–4 days shorter, in pigs challenged with the high compared to low dose. Interestingly, among pigs challenged with the high dose, the continuous and intermittent shedding states were on average up to 10–17 and 3–4 days longer, respectively, in pigs infected with S. Cubana compared to the other three serotypes. Pigs challenged with the high dose of S. Typhimurium or S. Derby seroconverted on average up to 8–11 days faster compared to the low dose. These findings highlight that Salmonella fecal shedding and immune response following Salmonella challenge are dose- and serotype-dependent and that the detection of specific Salmonella strains and immune responses in pigs are time-sensitive.Despite the public health importance of  Salmonella contaminated pork products Salmonella detection measures are critical for efficient Salmonella control in pigs, that will eventually decrease Salmonella exposure and infection risks to humans.Salmonellosis is a major public health burden that contributes to the significant economic cost worldwide Salmonella infection in pigs is frequent, it is seldom associated with clinical disease Salmonella in pigs is characterized by variable durations and an intermittent shedding pattern, that is discontinuous  excretion of the pathogen from the host. Asymptomatic infection and intermittent shedding pose a great problem in the detection and control of Salmonella in pigs. Therefore, immune response to Salmonella infection is often used to screen for Salmonella infection in pigs Salmonella in live pigs, it is critical to better understand the duration and dynamics of intermittent Salmonella fecal shedding and immune response post exposure and during infection, together with the factors that affect these processes.Although Salmonella serotype and dose affect the infection process. Specifically, it is reported that the dose of exposure affects fecal shedding Salmonella serotypes Salmonella serotypes at different doses may show different patterns and dynamics of Salmonella fecal shedding and immune response. Such information may have important implications in aiding the early detection of Salmonella in pigs, leading to its more effective control at the farm level. Detailed data on infection dynamics are also essential for further work on mathematical modeling of Salmonella transmission and intervention strategies.Previous studies suggest that Salmonella doses and serotypes tested  or high (0.65×109 CFU) dose of one of the four serotypes of Salmonella enterica subsp. enterica: Yoruba, Cubana, Typhimurium, or Derby . There were 8 serotype-dose combinations and 6 pigs in each serotype-dose group, resulting in a total of 48 pigs included in the dataset.For the purpose of this study, the datasets from Salmonella serotypes. Among pigs challenged with the high dose, 2 pigs in each group were monitored for an additional 2 weeks. Pigs were sampled once before challenge to confirm their infection-free status. Furthermore, they were sampled every day from the day of challenge (day 0) to day 5, and every 1–4 days thereafter. A total of 1,215 fecal samples were collected during the study. Salmonella detection was based on microbial culture with modified semi-solid Rappaport Vassiliadis (MSRV) enrichment, reported to have sensitivity and specificity of detecting Salmonella in pig fecal samples of 95% and 100%, respectively, and detection limit of 10 CFU/25 gram sample Salmonella serotype in feces of a pig on a given day.Pigs were monitored for 8 weeks for fecal excretion of the four Salmonella” from IDEXX Laboratories for S. Derby and S. Cubana  and “Svanovir” from Svanova Biotech for S. Typhimurium ). An in-house ELISA test was used to detect serum antibodies to S. Yoruba (O antigen 16). The results of ELISA tests were in the form of the level of antibodies detected in a pig on a given day.Blood samples were collected once a week for 8 weeks starting from day 0 of the experiment. Among pigs challenged with the high dose, 2 pigs were selected from each serotype group and their blood samples were collected once a week for the additional 2 weeks. A total of 446 serum samples were collected during the study period. To detect serum antibodies, commercial ELISA tests were used: “Herdcheck Swine Salmonella in feces and the level of immune response in pigs' sera during infection. Furthermore, the dataset provides information on the patterns of shedding and immune response over the course of infection which could be translated into allowed states and transitions in the corresponding multistate models. Specifically, it is easy to comprehend the difference in the meaning of a positive or negative test result depending on the elapsed time from the day of challenge that a particular sample of feces or blood was collected and examined. For example, absence of Salmonella from feces has a different meaning on the first day after challenge, in the middle of Salmonella infection and at the end of the infection, possibly indicating latency, intermittent non-shedding and recovery, respectively (which we define in the next paragraph).The longitudinal nature of the pig dataset provides information on the presence or absence of Salmonella excretion during infection reported in Salmonella. Thus, the observed intermittent shedding cannot be adequately explained by considering a monotonic decline in the shedding level following challenge. Considering sensitivity, specificity and detection limit of MSRV, it is reasonable to assume that the observed culture results represent the true status of animals with a reasonable degree of accuracy. Thus, combining the information on the culture result and its timing from the day of challenge, several facts were observed for fecal shedding in the pig dataset. Salmonella was never detected post challenge in feces of some pigs. Not all pigs started shedding immediately post-challenge. When pigs finally started shedding post-challenge, they may shed continually  for a variable period of time. After this period, some pigs were never found shedding again, while shedding could be detected on and off for variable short periods of time in other pigs. In the dataset, some of these latter pigs completely stopped shedding after some time while others continued this intermittent pattern of shedding until the study was terminated. To reflect these observations, the pig dataset was interpreted and states were defined as follows:The semiquantitative data on the level of 1 =  latency: negative sample on the day of and immediately after challenge;2 =  continuous shedding state: an uninterrupted sequence of positive samples after challenge;3 =  intermittent non-shedding state: negative samples following state 2 or state 4 if they were followed by positives before the end of the experiment;4 =  intermittent shedding state: positive samples following state 3;Salmonella from feces. Recovery following latency (state 1) indicates that the pig was never found to shed Salmonella in feces, suggesting that it did not become infected after exposure . These conditions for recovery were chosen to increase specificity in classifying pigs as recovered and to prevent false classification of positive pigs as recovered;5 =  recovery: negative samples following state 1, 2, or 4 if there were at least 5 consecutive negatives during at least a 14-day period. Recovery following shedding states (states 2 and 4) represents clearance of Salmonella was not detected because the pig has recovered from infection or because it has been in the temporary non-shedding state. In other words, censoring means that the observed state is known only to be one of a particular set of states . Note that in contrast to the usual terminology of survival analysis, here it is the state which is considered to be censored, rather than the event time 99 =  censored state: if a sample was negative but conditions for state 5 were not met, it is unknown whether NA =  sample not collected/examined.i = 1, 2, 3, and 4) and one absorbing state (i = 5). The arrows in ijq) show which instantaneous transitions are possible between states.The considered multistate fecal shedding model is shown in S. Yoruba, and the cut-off was calculated by using the mean of the samples collected pre-inoculation, i.e., when pigs were Salmonella-negative, and by adding two standard deviations. According to this cut-off, 2 pigs would be unrealistically classified as seroconverted already on day 0 of the experiment. To correct for that, for the purpose of this study, a “custom” cut-off for the in-house ELISA was defined as 1.5× of the highest antibody level detected on day 0. This was done to raise the specificity for detection of antibodies to S. Yoruba in order to avoid possibly false positive classifications in the model. For consistency in detecting antibodies to other Salmonella serotypes in the study, we applied the same custom cut-off in classification of the other ELISA tests results. However, because the use of a custom cut-off might have reduced the specificity of the commercial ELISA kits, we also ran the analysis using the data where the manufacturer recommended cut-offs were used to classify S. Cubana, S. Derby, and S. Typhimurium, while the custom cut-off was used to classify S. Yoruba ELISA data.The level of antibodies detected in a pig's serum on a given day was dichotomized using a cut-off so that the pig could be classified as having an immune response below or above the seroconversion level on a given day. The individual variation in the humoral immune response rules out the possibility to perfectly divide animals into these two groups. In the commercial ELISA kits, the validation of the cut-off has been performed by testing a large number of pig reference sera. In the Salmonella challenge. Some pigs did not seroconvert post challenge. In other pigs, the antibody level rose post-challenge at variable rates. In some pigs that seroconverted, the detected antibody level temporarily fell below the cut-off. Accordingly, negative and positive blood samples were coded with 1 and 2 indicating immune response below and above the seroconversion level, respectively. The considered multistate model for immune response (i = 1 and 2). The instantaneous transitions from state to state in the model shown in ijq.Combining the information on the serological test result and its timing from the day of challenge, the following was observed for pigs' immune responses to response  includesThe reader is directed elsewhere to obtain information about the theory underlying the multistate models e.g., . In the In the pig dataset, the sampling times were assumed to be non-informative, observation times were fixed, and the transition times were not exact. The effect of serotype and dose on the dynamics of fecal shedding and immune response was quantified by examining their effect on (i) the hazard ratios, (ii) ratios of transition intensities when more than one transition out of a state is possible, and (iii) the time spent in each state .0). Model selection involved forward selection of covariates associated significantly with the transition intensities. The best model was selected based on the Likelihood ratios (LR) and Akaike's Information Criterion (AIC). In such a saturated model the effect of the covariate differs in each of the allowed transitions  the transition times from each state are independent of the history of the process prior to entry to that state (the Markov assumption), 2) transition intensities are homogeneous across the pig population, and (3) transition intensities are homogeneous through time. Assumption (1) is inherent to many multistate models; it could not be tested due to absence of data on the exact transition times  transitii to state j and the observed total time spent in state i  prevalences of pigs in different states. CI were estimated by simulating 1,000 random vectors from the asymptotic multivariate normal distribution implied by the maximum likelihood estimates (and covariance matrix) of the log transition intensities and covariate effects, then calculating the expected prevalences for each replicate Salmonella was detected in 532 out of 1,215 fecal samples. Of these positive samples, 344 denoted the presence of a pig in the continuous shedding state and 188 in the intermittent shedding state. There were 683 negative samples, of which 56, 202, and 371 were classified into the latent, intermittent non-shedding and recovered states, respectively, while 54 samples were censored . There were 304 observations each of S. Cubana, S. Typhimurium, and S. Derby, and 303 of S. Yoruba. For the low and high challenge doses, there were 575 and 640 observations, respectively. Initial values of parameters in the shedding model were 12q = 0.75, 15q = 0.11, 23q = 0.06, 25q = 0.01, 3q4 = 0.16, 43q = 0.10, and 45q = 0.03.In the pig dataset, S. Cubana were used as the reference groups. Compared to the H0 model without covariates (AIC = 1021.2), these single-covariate analyses indicated a statistically significant association between the model transition intensities and serotype with constraints 15q = 25q = 45q = −23q = 43−q, meaning an equal effect of serotype on all recovery transitions, i.e., 15q, 25q, and 45q, and equal effect but in opposite direction on the transitions to the intermittent non-shedding state, i.e., 23q and 43q  = 16-7 = 9, χ2 p-value = 0.01; AIC = 1017.6). The dose was found to be associated with transition intensities in the saturated form . Multivariate modeling results further indicated that the best model for fecal shedding dynamics includes both the challenge serotype (in the above described constrained form) and dose (in the saturated form). A comparison of the observed and model-predicted prevalences with 95% CI for this model indicated a reasonably good fit to the data used for its development and so supported the internal validity of the model , from the continuous shedding state to recovery , and from the intermittent shedding state to recovery . That means that pigs challenged with the high dose tend to spend more time shedding both as part of the continuous and intermittent shedding states compared to those challenged with the low dose. Compared to pigs infected with S. Cubana, pigs infected with S. Typhimurium or S. Derby had significantly reduced risks of recovery , both having HR = 0.4 . On the other hand, the risk of transition to intermittent non-shedding either after the continuous or intermittent shedding, was significantly higher in pigs infected with S. Typhimurium  or S. Derby , compared to S. Cubana. Taken together, this means that pigs challenged with S. Cubana are less likely to become infected compared to S. Typhimurium and S. Derby. However, when they do become infected they will tend to go through longer continuous and intermittent shedding states after which they will be more likely to recover than S. Typhimurium and S. Derby (which will tend to transition between the intermittent shedding and non-shedding states more often with a short period of occupancy of these states at each visit). Therefore, the total duration of S. Cubana infection will likely be shorter compared to S. Typhimurium and S. Derby. There was no evidence of the statistically significant effect of dose and serotype on the other model transitions. Likewise, there was no evidence of any difference between S. Cubana and S. Yoruba.From the final fecal shedding model, we estimated hazard ratios (HR) and the corresponding 95% CI. Compared to pigs challenged with the low dose of S. Typhimurium. Thus, it is evident from 9) of S. Typhimurium Salmonella continuously until day 10 post-inoculation and that shedding became intermittent thereafter. The period during which all pigs shed Salmonella can be regarded as the minimum duration for which all pigs remain in the continuous shedding state. Thus, the finding by S. Typhimurium . Moreover, the 8 CFU of S. Typhimurium and tested for fecal shedding every 1 or 2 weeks for 108 days, shedding was detected up to 92 days post-inoculation To test the generalizability of our findings, we compared the model outputs with the available literature. In their experimental inoculation studies with a high dose , the number of samples classified below and above the seroconversion level was 273 and 173, respectively, and in the corresponding multistate model one of the initial values was slightly different (12q = 0.02).Of 446 serum samples in the pig dataset, using the custom cut-off, 196 and 250 samples were classified as below and above the seroconversion level, respectively. In the corresponding multistate model, the initial values were S. Yoruba were used as the reference groups. The results described below pertain to the model that used ELISA data dichotomized using the custom cut-offs except when it is explicitly stated that the manufacturer recommended cut-offs were used for the commercial ELISA kits.In the analysis of the immune response multistate model, the low challenge dose and 12q = −21q). In other words, each of the covariates showed equal but opposite effects on the progression to seroconversion and regression to the antibody level below the seroconversion level. From comparison with the model without covariates , statistics were as follows for the serotype: LR = 51.0, df = 5-2 = 1, χ2 p-value <10−10; AIC = 240.2, and for dose: LR = 6.4, df = 3-2 = 1, χ2 p-value  = 0.01; AIC = 280.8. The results of multivariate modeling indicated that the best model for immune response post-challenge has both the dose and serotype included in the above described constrained form (12q = −21q). There was no evidence of interaction between the two considered covariates and time heterogeneity in the models.The single-covariate analyses indicated a statistically significant association between model transition intensities and each of the covariates, with both covariates being best represented in the reduced form such that the covariate effect is in the opposite direction between the progression and regression transitions , HR = 18.1 , and HR = 4.5 , respectively. Likewise, S. Typhimurium, S. Derby, and S. Cubana had HR = 0.1 , HR = 0.1 , and HR = 0.3 , respectively, for returning to the “below seroconversion level” state. In other words, S. Typhimurium and S. Derby, and to a lower extent S. Cubana, are expected to seroconvert much faster and remain above the seroconversion level much longer than S. Yoruba.HRs estimated for this model indicated that pigs infected with the high dose had a statistically significantly higher risk of seroconverting  and a lower risk of transitioning back to the “below seroconversion level” state  than those challenged with the low dose. In other words, we can expect that pigs challenged with the high dose will seroconvert faster and remain above the serocoversion level longer. The effect of serotype on the risk of seroconversion was even more pronounced; compared to Salmonella serotypes, we also ran the analyses using the available manufacturer recommended cut-offs for the commercial ELISA kits and the custom cut-off for the in-house ELISA (used for S. Yoruba). According to the manufacturer recommended cut-off, none of the 12 pigs challenged with S. Cubana seroconverted post-challenge. Thus, S. Cubana data were excluded from this analysis. The covariates and constraints in the best model were identical to the one developed for the model using custom cut-offs. The estimated HRs indicated similar albeit weaker effects of covariates. For high dose vs. low dose, seroconverting and transition to the “below seroconversion level” state had HR = 2.6  and HR = 0.4 , respectively. Compared to S. Yoruba, the other two strains, S. Typhimurium and S. Derby, had respectively HR = 7.2  and HR = 7.7  for seroconverting, and respectively HR = 0.1  and HR = 0.1  for transitioning back to the “below seroconversion level” state.To evaluate the effect of using the custom cut-off for all S. Cubana and S. Yoruba could not be accurately estimated because some pigs never seroconverted. Similarly, the times spent above the seroconversion level could not be accurately estimated because the experiment was not run for a long enough time to capture waning of immunity in pigs.S. Typhimurium, seroconversion has been detected as early as 16 days post-inoculation with the majority of pigs, with 94% (15 of 16) seroconverting between the third and fifth week (days 22 and 39) post-inoculation 8 of S. Typhimurium started seroconverting at day 7 post-inoculation with the majority of pigs seroconverting by day 22 7 or 3.2×109 and tracer pigs housed with them seroconverted within 1–3 weeks post-inoculation S. Typhimurium  estimated using the manufacturer recommended cut-offs for the commercial ELISA kits.Both custom and manufacturer cut-off based models showed good internal validity . To asseSalmonella challenge dose (high v. low) and four serotypes  on the dynamics of Salmonella fecal shedding and immune response post-challenge. We found strong evidence that the patterns and dynamics of fecal shedding and immune response are significantly and meaningfully affected by both the serotype and dose of exposure. Below we discuss these findings in the context of existing research needs and knowledge, and we elaborate on the associated implications and limitations.In this study, we used a multistate Markov modeling approach to evaluate the effect of Salmonella into the environment that can eventually be transmitted to other animals and humans. Of particular concern is that Salmonella and many other infectious agents, such as enterohemorrhagic Escherichia coli and Listeria monocytogenesSalmonella in feces, the individual is not infectious to other members of the population. On the other hand, such infected individuals are not detected by fecal culture. It is therefore critical to understand the ecology and selective pressures governing intermittent fecal shedding so that intermittently-shed pathogens can be controlled more effectively. This highlights the value of the results of our study discussed below.Fecal shedding represents an important mechanism of spreading Salmonella fecal shedding multistate model performed in this study indicated that pigs challenged with the high dose of 109 CFU of Salmonella on average started to shed Salmonella in feces up to 1.5 day sooner and, depending on the serotype, had 10–26 days longer shedding during the continuous shedding state compared to those challenged with the low dose (106 CFU) of Salmonella. Also, intermittent non-shedding was on average 2–4 days shorter in pigs challenged with the high compared to low dose. Therefore, reduction of the dose of exposure  would shorten the periods of shedding and extend the periods of intermittent non-shedding and would, consequently, decrease the spread of the infection through the population.The analyses of the S. Cubana had continuous and intermittent shedding states on average up to 10–17 and 3–4 days longer, respectively, than the other three evaluated serotypes, of which S. Yoruba had shedding times most similar to S. Cubana. The similarity between S. Yoruba and S. Cubana was also supported by the lack of statistically significantly different HRs comparing their model transition intensities. Our results also indicated that compared to S. Yoruba and S. Cubana, pigs infected with S. Typhimurium and S. Derby are far more likely to enter the intermittent non-shedding state following the continuous or intermittent shedding states than to recover from these states. Overall, these findings suggest that pigs infected with S. Typhimurium and S. Derby tend to shed Salmonella in shorter blocks of time (both as part of the continuous and intermittent shedding states) compared to S. Yoruba and S. Cubana. However, S. Typhimurium and S. Derby are more likely to (re-)enter the intermittent non-shedding state. Consequently, they showed a tendency for longer overall duration of host infection with similar or longer total duration of fecal shedding  compared to S. Yoruba and S. Cubana  and the less invasive S. Yoruba and S. Cubana (which are mostly associated with contaminated feed). The challenge in controlling such infections is that for the invasive classical pig serotypes, ceasing or reducing pathogen replication and consequently shedding hampers their detection while seemingly it also causes the pathogen to reside in the host longer, which extends the period of time a pig is infected and thus capable of transmitting the pathogen to another host. The serotypes associated with contaminated feed are also of concern because through their feed-borne spread, many herds may become infected in a short time, and this route of transmission can therefore be regarded as a major threat to the control of Salmonella in pigs. Moreover, if there is no control of Salmonella in feed, pigs may be continuously exposed via feed, thereby producing a different shedding pattern than with only a single exposure. Indeed, the high significance of commercial feed as a potential vehicle of Salmonella transmission has been reported in the USA S. Cubana has been isolated from routine environmental samples from feed mills in Sweden S. Yoruba was detected in a Swedish specific pathogen-free pig herd, feed was concluded to be the most likely source S. Cubana were reported in 1998, affecting 34 and 14 people Salmonella enterica are considered a potential threat to human health Our study also indicated that pigs infected with . Cubana . This trSalmonella in fecal samples has good diagnostic performance , and its detection limit is around 10 CFU/25 g feces. The number of false negatives should thus be fairly low, while there should be no false positives due to the test. The semiquantitative results for the level of Salmonella in feces of individual pigs in the experimental studies used here Salmonella. This, together with the MSRV's diagnostic performance and the detection limit, supports the finding that the intermittent non-shedding state truly occurs as opposed to representing blocks of false negative classifications. Also, it is unlikely that we would observe just by chance the inverse relationship between the strain-specific duration of a one-time stay in the fecal shedding states and the total duration of infection and shedding. Therefore, misclassification bias does not seem to be of high concern in this study. Any misclassification would be rather in the form of assuming non-shedding instead of low-shedding. The conclusions and the biological relevance would remain the same, as low-shedding (below the detection limit) would have more or less the same effect on detection and spread as non-shedding. Nevertheless, a formal assessment of this question could be done through an extension of the developed model into a Hidden Markov model in a future study.The findings of this study are obviously dependent on the correct classification of pigs into one of the possible shedding and non-shedding states, the validity of the structure of the considered multistate model for fecal shedding , and the underlying assumptions. With respect to a possible misclassification bias, a Salmonella and many other pathogens, it has rarely been specifically considered in models of the within-host or among host-infection dynamics. The S. Typhimurium transmission within a pig herd. Similarly, the L. monocytogenes in feces. However, to our knowledge, the study described here is unique in the way it explicitly separated the shedder animals into those that are in the continuous and intermittent shedding states, which has important implications for infection detection and transmission, as already discussed. Because samples were not collected daily, it is possible that some pigs were negative even before the first negative sample was obtained. Therefore, the duration of continuous shedding might have been overestimated. However, given the fairly similar frequency of sample collection before and after the first negative sample was collected, and the favorable validation with While intermittent shedding is widely known to occur during infection with Salmonella infection among animals housed together during the experiment. Such transmission would result in super-infections, re-infections  or passive shedding of the ingested Salmonella, all of which would cause underestimation of the duration of non-shedding states and overestimation of the duration of shedding states. We cannot rule out these possibilities. However, the first two possibilities seem unlikely because some pigs developed antibodies to Salmonella which presumably have had a protective effect. Moreover, experimental exposure of naïve pigs to shedding pigs has shown that the transmission rates between pigs are low Our model of the within-host dynamics of fecal shedding assumes a single challenge at day 0. It completely ignores the possibility of pig-to-pig transmission of S. Typhimurium or S. Derby seroconverted 8 to 11 days faster than those infected with the low dose of these serotypes. Our analyses also indicated that these estimates strongly depend on the chosen manufacturer or custom derived cut-offs of the seroconversion level. Specifically, using manufacturer as opposed to custom cut-offs resulted in a longer estimated time to seroconversion for S. Typhimurium and S. Derby: approximately 5 days (or 30%) in pigs challenged with the low dose and approximately 3 days (or 50%) in pigs challenged with the high dose. Seroconversion to S. Cubana and S. Yoruba was approximately 4 and 14 times slower, respectively, than seroconversion to S. Typhimurium and S. Derby in pigs infected with the high dose. When manufacturer cut-offs were used, seroconversion to S. Yoruba was 7 times slower than that estimated for S. Typhimurium and S. Derby. That may be because S. Typhimurium and S. Derby are more invasive than S. Cubana and S. Yoruba and therefore invade the intestinal epithelium to a greater extent and induce antibody secretions more rapidly, in higher amounts, and over longer time periods than the feed-associated serotypes. Because pigs infected with the low dose or with S. Cubana or S. Yoruba need more time to seroconvert, more time would pass before the infection could be detected in the infected herd. While the custom cut-off has not been validated and so its validity is unknown, it is reasonable to speculate that for the tested serotypes it has a very good sensitivity but questionable specificity (except for the custom cut-off for S. Yoruba which was set so to achieve better specificity) and so may have a considerable number of false positive classifications. As a consequence, the time to seroconversion might be underestimated for S. Typhimurium, S. Derby, and S. Cubana in the model which used the custom cut-offs. On the other hand, it is reasonable to speculate that the manufacturer-recommended cut-offs might have a superior specificity at the cost of a reduced sensitivity to reduce the possibility of false positive classifications. Under these circumstances the model might have overestimated the time to seroconversion. The custom cut-off was useful in our setting to detect immune responses in pigs that were known to be exposed. It would be less useful in the field when the aim is to detect infected animals and the true status of exposure is unknown. Furthermore, test specificity is particularly important in societies where Salmonella control policy imposes mandatory restrictions and/or interventions in infected herds (such as movement restrictions of positive herds and euthanasia of positive pigs).The performed analysis of immune responses indicated that pigs infected with the high dose of Salmonella fecal shedding and immune responses in pigs over a period of time based on frequent enough sample collection and testing. Nevertheless, the available information L. monocytogenes fecal shedding using a Markov Chain modeling approach Salmonella fecal shedding and immune response and identify interesting new insights in the relationship between shedding episodes and total length of infection and shedding.There are limited experimental studies reporting L. monocytogenes in cattle Salmonella and the effects of different intervention and/or prevention measures.Our study could be used to suggest new avenues for research. For example, we emphasize the need to perform long enough longitudinal studies of fecal shedding and immune response to quantify the duration of fecal shedding states and immunity, particularly for high infection doses and classical pig serotypes. Since stress has been speculated to affect fecal shedding in pigs Salmonella challenge based on which new insights were possible. These results contributed to an understanding of the epidemiology of salmonellosis in pigs, demonstrating that the Salmonella serotype and the dose of exposure have profound effects on the pattern and duration of fecal shedding and host immune response. Such understanding may help improve the screening of different Salmonella serotypes in the pig reservoir, thereby decreasing the risk of human infection.In conclusion, the results of multistate modeling described in this study provided quantification and formal assessment of the previously described natural course of fecal shedding and immune responses post


Identifying the targets of broadly neutralizing antibodies to HIV-1 and understanding how these antibodies develop remain important goals in the quest to rationally develop an HIV-1 vaccine. We previously identified a participant in the CAPRISA Acute Infection Cohort (CAP257) whose plasma neutralized 84% of heterologous viruses. In this study we showed that breadth in CAP257 was largely due to the sequential, transient appearance of three distinct broadly neutralizing antibody specificities spanning the first 4.5 years of infection. The first specificity targeted an epitope in the V2 region of gp120 that was also recognized by strain-specific antibodies 7 weeks earlier. Specificity for the autologous virus was determined largely by a rare N167 antigenic variant of V2, with viral escape to the more common D167 immunotype coinciding with the development of the first wave of broadly neutralizing antibodies. Escape from these broadly neutralizing V2 antibodies through deletion of the glycan at N160 was associated with exposure of an epitope in the CD4 binding site that became the target for a second wave of broadly neutralizing antibodies. Neutralization by these CD4 binding site antibodies was almost entirely dependent on the glycan at position N276. Early viral escape mutations in the CD4 binding site drove an increase in wave two neutralization breadth, as this second wave of heterologous neutralization matured to recognize multiple immunotypes within this site. The third wave targeted a quaternary epitope that did not overlap any of the four known sites of vulnerability on the HIV-1 envelope and remains undefined. Altogether this study showed that the human immune system is capable of generating multiple broadly neutralizing antibodies in response to a constantly evolving viral population that exposes new targets as a consequence of escape from earlier neutralizing antibodies. Four sites of vulnerability for broadly neutralizing antibodies to HIV-1 have been identified thus far. How these broadly reactive antibodies arise, and the host-pathogen interactions that drive the affinity maturation necessary for neutralization breadth are poorly understood. This study details the sequential development of three distinct broadly neutralizing antibody responses within a single HIV-1 infected individual over 4.5 years of infection. We show how escape from the first wave of antibodies targeting V2 exposed a second site that was the stimulus for a new wave of glycan dependent broadly neutralizing antibodies against the CD4 binding site. These data highlight how antibody evolution in response to viral escape mutations served to broaden the host immune response to these two epitopes. Finally, we document a third wave of neutralization that targets an undefined epitope that did not appear to overlap with the four known sites of vulnerability on the HIV-1 envelope. These data support the design of templates for sequential immunization strategies aimed at increasing neutralization breadth through the recognition of multiple epitopes and their immunotypes. Functionally conserved sites are protected by extensive glycosylation, and large solvent exposed hypervariable structures  Neutralizing antibodies are the principal correlate of protection for most preventative vaccines. Designing suitable vaccine immunogens to elicit these types of antibodies has been relatively simple for conserved pathogens such as smallpox and other DNA viruses. For more diverse pathogens like HIV-1, the neutralizing antibodies elicited by vaccination or during natural infection are largely strain-specific and therefore would not be protective against globally circulating viral variants Plasma mapping strategies and the isolation of monoclonal antibodies have defined four major targets for broadly neutralizing antibodies on the HIV-1 glycoprotein In the CAPRISA 002 Acute Infection Cohort, we previously identified seven individuals with broadly neutralizing antibodies. In five cases we were able to map the plasma antibody specificities to known epitopes  We have previously described the development of neutralization breadth in CAP257 using longitudinal plasma samples from HIV-1 seroconversion to three years post-infection (p.i.) The first wave of neutralization breadth (typified by CAP63) peaked at 67 weeks p.i. with a maximum titer of 1∶1,493 and exclusively neutralized subtype C viruses . We adsorbed out the gp120 binding antibodies in plasma samples from the peak neutralizing activity of each wave using recombinant ConC gp120 coupled to tosyl-activated magnetic beads . The adsThe inability of gp120 to adsorb out wave 1 neutralization suggested these antibodies might recognize the trimer specific epitope in V1/V2 defined by PG9/16 To define whether the V2 epitope recognized by CAP257 plasma antibodies overlapped with that of known broadly neutralizing antibodies to this site, we tested the sensitivity of PG9/16, CH01-04, and PGT145 to the same ConC V2 mutations described above and compared them to CAP257 neutralizing antibodies at the peak of wave 1 activity, 67 weeks p.i. . Of the To define escape from wave 1 neutralizing antibodies, we examined sequences from the V2 region of CAP257 over time. Using single genome amplification (SGA) we obtained 125 full envelope sequences from twelve time points between 7 and 213 weeks p.i., and focused on the N160 glycan and the cationic C-strand in V2 that are the targets of wave 1 antibodies . Interess), was then compared to the parental CAP257 3 yr virus using longitudinal plasma samples. In contrast with the resistant CAP257 3 yr virus, the back-mutated V1/V2 virus (CAP257 3 yr(V1/V2s)) became sensitive to neutralization at 23 weeks p.i. (To test this we selected an envelope from 174 weeks p.i. (CAP257 3 yr) that was completely resistant to CAP257 neutralizing antibodies , and back-mutated the V1/V2 region to match the earliest sequence from 7 weeks p.i. . The neus) back-mutated envelope. To establish whether these strain-specific V1/V2 neutralizing antibodies targeted the same epitope as wave 1 broadly neutralizing antibodies, we introduced selected escape mutations  into the sensitive CAP257 3 yr in the CAP257 3 yr(V1V2s) virus conferred slight neutralization sensitivity coinciding temporally with wave 2 .The V1/V2 sub-domain of gp120 plays an important role in shielding the envelope from neutralization. More specifically, several modifications in V1/V2 (particularly at N-glycosylation sites) have been implicated in either shielding or exposing the CD4bs to neutralizing antibodies To further characterize the epitope targeted by wave 2 antibodies, we assessed the dependence of wave 2 binding on the D368 residue in α3 , and residues 474/475/476 in α5  by adsorption studies. The D368R or D474A/M475A/R476A mutations were separately introduced into ConC gp120, and compared to the wild-type protein for their ability to adsorb out the neutralizing activity against Q842 and RHPA at peak wave 2 titers. Both mutant gp120s  with the effects of two alanine substitutions  in the N276 glycosylation sequon . The N2750  and VRC01 (CD4bs). Both monoclonal antibodies were profoundly affected by the R456W mutation (514 and 30 fold respectively). Like CAP257, HJ16 neutralization was significantly dependent on the glycan at position 276 with glycan deleting mutations (N276Q/A and T278A) resulting in a 74–106 fold increase in IC50 . This deTo clarify the role of glycan binding we tested three CD4bs neutralizing antibodies  in ELISA for binding to either the glycosylated or deglycosylated RHPA gp120 core proteins . The neuWhile the simultaneous introduction of T278A, N279D, and R456W mutations into heterologous viruses Q842 and RHPA made them resistant to wave 2 neutralization at 122 weeks p.i. , when eaTo better characterize their contributions to escape from wave 2 antibodies, each mutation  was introduced separately into Q842 and RHPA and compared to the wild-type viruses. The N279D mutation , and those first neutralized at 93 weeks p.i.  after the emergence of initial wave 2 escape mutations . VirusesLike wave 1, wave 3 neutralization could not be adsorbed with monomeric gp120, suggesting that these antibodies targeted a quaternary epitope, or an epitope in gp41. To assess whether wave 3 was a distinct antibody specificity, or a re-emergence of wave 1 antibodies, we selected a virus (Du156) that was sensitive to waves 1 and 3, and less sensitive to wave 2  with a hundred fold drop in titer Wave 2 antibodies targeted a known site of vulnerability, the CD4bs, but these antibodies had an unusual glycan dependent mechanism of neutralization. CAP257 wave 2 and HJ16 neutralization were both highly dependent on interactions with the N276 glycan. N276 is also the recently described target of the broadly neutralizing antibody 8ANC195, but this antibody does not appear to interact with the CD4bs Characterization of viral escape from CAP257 CD4bs antibodies indicated that deletion of the N276 glycan alone did not confer complete resistance. Escape required accumulating mutations in the CD4bs site, consistent with the functional conservation of this epitope. In addition to deletion of the N276 glycan, CAP257 escape occurred through a R456W mutation that also significantly affected neutralization by VRC01 (30 fold) and HJ16 (514 fold). This mutation likely contributed towards the evolution of VRC01 resistant virus by 174 weeks. W456 is extremely rare, occurring in only 0.78%  of sequences in the LANL HIV-1 sequence database. A crystal complex for HJ16 is not available, however the structure of VRC01 bound to its epitope showed that this antibody does not make significant contact with the R456 side chain in gp120, but rather hydrogen bonds with the R456 backbone carbonyl group. This suggests that the R456W mutation provides an indirect mechanism for resistance. The highly conserved R456 side chain can make hydrogen bonds with backbone carbonyl groups of amino acids at position 277 and 278 in the D-loop, as well as hydrogen bonds with E466 side chain in β24, C-terminal to V5 . Loss ofThis study adds to data showing that the immune system can target multiple conserved epitopes et al. showed that two light chain variants paired with a single heavy chain of a strain-specific neutralizing antibody differentially neutralized early autologous envelopes Sequential immunization may be a useful strategy to promote the broadening of the B-cell response. Recently Murphy Overall these data highlight how interactions between the host immune system and viral escape mutations shaped the development of broadly neutralizing antibodies. The escape pathways identified here that led to the increased breadth of neutralization for both V2 and CD4bs antibodies provide potential pathways for generating broadly neutralizing antibodies. Further defining these pathways through the isolation of monoclonal antibodies will provide valuable insight into how these types of antibodies could be elicited using sequential immunization in a vaccine setting.The CAPRISA Acute Infection study received ethical approval from the Universities of KwaZulu-Natal (E013/04), Cape Town (025/2004), and the Witwatersrand (MM040202). CAP257 provided written informed consent for study participation.The CAPRISA Acute Infection cohort is comprised of women at high risk of HIV-1 infection in Kwa-Zulu Natal, South Africa env sequences were assembled and edited using Sequencher v.4.5 (Genecodes) and Bioedit v.7.0.5.3.The amplification of envelope genes from single HIV-1 RNA genomes has been previously described 2 in DMEM containing 10% heat-inactivated fetal bovine serum (Gibco BRL Life Technologies) with 50 ug/ml gentamicin (Sigma) and disrupted at confluency by treatment with 0.25% trypsin in 1 mM EDTA (Sigma).The TZM-bl cell line engineered from CXCR4-positive HeLa cells to express CD4, CCR5, and a firefly luciferase reporter gene (under control of the HIV-1 LTR) was obtained from the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH  with PfuUltra II (Stratagene), purified by a Gel Extraction Kit (QIAGEN) and cloned into pcDNA3.1 (Invitrogen). The envelope plasmids were co-transfected into 293T cells using FuGENE 6 (Roche) with the pSG3ΔEnv backbone . Cultures were incubated for 48 hours to produce Env-pseudotyped viral stocks that were filtered through 0.45 µm and frozen in DMEM supplemented with 20% FBS. Mutant envelopes were generated with the QuikChange Lightning Kit (Stratagene) and confirmed by DNA sequencing (as above).50 calculated as the dilution at which the infection was reduced by 50%.The TZM-bl neutralization assay has been described previously Plasmids encoding Histidine tagged recombinant envelope proteins were transfected into 293T cells using polyethylenimine 25 kDa (Polysciences). Recombinant proteins were expressed and purified as previously described 2SO4. Absorbance was read at 450 nm on a VERSAmax tunable microplate reader (Molecular devices).Protein antigens were coated at 4 µg/mL onto high binding 96-well ELISA plates (Corning) overnight at 4°C. All subsequent steps were carried out in 5% fat-free milk, 0.05% Tween20 in PBS for 1 hour at 37°C. The plates were blocked and then probed with serial dilutions of the adsorbed plasma or specific monoclonal antibodies, biotinylated goat anti-human polyclonal antibodies (KPL), and an anti-biotin monoclonal conjugated to HRP . Antigen-antibody complexes were detected by incubating with 100 µL 1-Step Ultra TMB-ELISA (Thermoscientific) for five minutes and then the reaction was stopped with 25 µL 1 M HPlasmid encoding the RHPA gp120 core was transfected with polyethylenimine-MAX 40 kDa (Polysciences) into GnTI(−/−) 293S cells and purified using two step lectin chromatography and Ni-NTA affinity chromatography. Protein was assessed for purity and conformation by SDS-PAGE and ELISA. 1 mg of the RHPA core gp120 was deglycosylated overnight at 37°C in 500 mM sodium chloride, 100 mM sodium acetate pH 5.5 with 0.5 U of Endo-H. Glycans were removed through buffer exchange into PBS using Vivaspin 20 mL concentrators (Sartorius stedim). Deglycosylation was confirmed by SDS-PAGE and sandwich ELISA (described above) using lectins as a capture protein.


RP1) gene are a common cause of autosomal dominant retinitis pigmentosa (adRP), and have also been found to cause autosomal recessive RP (arRP) in a few families. The 33 dominant mutations and 6 recessive RP1 mutations identified to date are all nonsense or frameshift mutations, and almost exclusively (38 out of 39) are located in the 4th and final exon of RP1. To better understand the underlying disease mechanisms of and help develop therapeutic strategies for RP1 disease, we performed a series of human genetic and animal studies using gene targeted and transgenic mice. Here we report that a frameshift mutation in the 3rd exon of RP1  found in a patient with recessive RP1 disease causes RP in the homozygous state, whereas the heterozygous carriers are unaffected, confirming that haploinsufficiency is not the causative mechanism for RP1 disease. We then generated Rp1 knock-in mice with a nonsense Q662X mutation in exon 4, as well as Rp1 transgenic mice carrying a wild-type BAC Rp1 transgene. The Rp1-Q662X allele produces a truncated Rp1 protein, and homozygous Rp1-Q662X mice experience a progressive photoreceptor degeneration characterized disorganization of photoreceptor outer segments. This phenotype could be prevented by expression of a normal amount of Rp1 protein from the BAC transgene without removal of the mutant Rp1-Q662X protein. Over-expression of Rp1 protein in additional BAC Rp1 transgenic lines resulted in retinal degeneration. These findings suggest that the truncated Rp1-Q662X protein does not exert a toxic gain-of-function effect. These results also imply that in principle gene augmentation therapy could be beneficial for both recessive and dominant RP1 patients, but the levels of RP1 protein delivered for therapy will have to be carefully controlled.Mutations in the retinitis pigmentosa 1 ( RHO) and CHM, were discovered two decades ago Recent reports of the success of Phase I clinical trials of gene therapy for Leber congenital amaurosis (LCA) and X-linked adrenoleukodystrophy (ALD) indicate that we are entering an era of genetic therapies, at least for some forms of genetic disease RP1 gene are the second most common cause of autosomal dominant RP , and have also been found to cause autosomal recessive RP (arRP) RP1 have been identified to cause adRP  and one pair of compound heterozygous mutations (c.5_6delGT and c.4941_4942insT)  [34], . InRp1-Q662X mRNA is spliced correctly and produces a truncated protein in photoreceptor cells, retinas from 4-week-old Rp1+/+, Rp1+/Q662X, and Rp1Q662X/Q662X mice were evaluated using RT-PCR, Western blot and immunofluorescence analyses. The RT-PCR results showed that mutant Rp1-Q662X allele was correctly spliced  of retinas from the Rp1Q662X/Q662X mice was two to three rows of nuclei thinner than that in wild-type controls. By 6 month of age, only 3–4 rows of photoreceptor nuclei remained in the Rp1Q662X/Q662X mice  and LAP (EGFP - S-tag) tags at the N-terminus of the Rp1 coding sequence in a BAC that contains 140 kb of genomic DNA surrounding Rp1 (Rp1 transgenic mice (designated T1–T3) and three lines of N-LAP-Rp1 transgenic mice (designated L1–L3) were produced following microinjection of each BAC into oocytes Rp1 or N-LAP-Rp1 transgenes to approximately half of their offspring.Expression of correctly localized truncated Rp1 protein in the retinas of the ding Rp1  [48], , [4−2 scot cd s m−2. The saturating rod a-wave was elicited by a white flash of 500 scot cd s · m−2 intensity produced by a xenon lamp (“High-intensity ERG” by the Standard). The single cone ERG was elicited by a 500 scot cd s · m−2 intensity white flash delivered on a 30 scot cd · m−2 background suppressing the rod activity. The ERGs were recorded in a 0.1 to 300 Hz bandwidth, digitized at 1 kHz, and the amplitudes of the respective responses were measured from the baseline to peak for the a-wave, and from the trough to peak for the rod and cone b-waves. Each trace is the average of 3 to 10 individual records . Statistical analysis was performed using Prism v3.02 software .Electroretinography (ERG) testing in the mice was performed as previously described RP1 transcript containing exon 2 to 3 and exon 3 to 4. The primer sets used were: forward, 5′-TCGGCCCTGGCTGAGTAGTCG-3′ (on exon 2), reverse, 5′-TTCCTGGTTTAAATGGCTCCCTTCC-3′ (on exon 3); and forward, 5′-TGCCAAGTTACCAGGAATCTC-3′ (on exon 3), reverse, 5′-CGACCGTCATCGTACCATCTTG-3′ (on exon 4).Retinas from animals of different genotypes at the desired ages were collected and homogenized in Trizol . Total RNA was extracted from one retina and reverse transcribed using Superscript II (Invitrogen). The PCR reactions were then performed using 2 µL of first-strand cDNA to amplify the region of Retinas from animals of different genotypes at the desired ages were collected and homogenized in sample buffer . Solubilized retinal protein samples were then processed for Western blot analysis as described Rp1-Q662X knock-in mice and transgenic mice were processed for immunostaining experiments as described previously Eyes from Figure S1Q662X/Q662X knockin mice.Expression of the full-length N-SF-TAP-Rp1 and N-LAP-Rp1 proteins prevents photoreceptor degeneration in Rp1A. Retinal histology from 12-month-old mice of the genotypes indicated. The retinal structure of the combined Rp1-Q662X : N-SF-TAP-Rp1 mice is essentially normal, in contrast to the early photoreceptor degeneration in the Rp1-Q662X mice. There is partial preservation of retina structure in the combined Rp1-Q662X : N-LAP-Rp1 mice . B. Ultrastructure of PSCs in 12-month old mice of the genotypes indicated. Note that the structure of the PSC and organization of the outer segment discs are normal in the combined Rp1-Q662X : N-SF-TAP-Rp1 mice, in contrast to the grossly disorganized PSC observed in the Rp1Q662X/Q662X mice. More significant disorganization of PSCs was observed in the combined Rp1-Q662X : N-LAP-Rp1 mice, including shorter outer segments with a mixture of normal discs and disorganized discs .(TIF)Click here for additional data file.


More than three billion populations are living under the threat of Japanese encephalitis in South East Asian (SEA) countries including India. The pathogenesis of this disease is not clearly understood and is probably attributed to genomic variations in viral strains as well as the host genetic makeup. The present study is to determine the role of polymorphism of TNF-alpha promoter regions at positions -238G/A, -308G/A, -857C/T and -863C/A in the severity of Japanese encephalitis patients.Total of 142 patients including 66 encephalitis case (IgM/RT-PCR positive), 16 fever cases (IgM positive) without encephalitis and 60 apparently healthy individuals (IgG positive) were included in the study. Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) using site specific restriction enzymes were implemented for polymorphism study of TNF alpha promoter.Following the analysis of the digestion patterns of four polymorphic sites of the TNF- alpha promoter region, a significant association was observed between the allele -308A and -863C with the patients of Japanese encephalitis.TNF- alpha 308 G/A has been shown to be associated with elevated TNF- alpha transcriptional activity. On the other hand, polymorphism at position -863C/A in the promoter region has been reported to be associated with reduced TNF- alpha promoter activity and lower plasma TNF levels. As per the literature search, this is the first study to identify the role of TNF- alpha promoter in JE infection. Our results show that subjects with - 308A and -863C alleles are more vulnerable to the severe form of JE infection. Japanese encephalitis (JE) is an important flaviviral disease of public health concern in Eastern and South-East Asian (SEA) countries. Approximately 50,000 cases are being reported annually to WHO with a case fatality of 10,000 cases ,3. In thTumor necrosis factor is a multifunctional pro-inflammatory cytokine with a role in modulation of acute inflammation and host innate immunity. Its association towards disease progression has well been observed in several acute and chronic infections and autoimmune diseases. The reactivation of latent tuberculosis following treatment with anti-TNF antibody (Infliximab) has been reported , indicatThe variations in the capacity to produce cytokines in different individuals have been attributed to the existence of polymorphisms within the regulatory regions or signal sequences of the cytokine genes. The present study aimed to associate the role of TNF-α promoter regions at positions -238, -308, -857 and -863 with disease progression and outcome in patients with Japanese encephalitis. The single nucleotide polymorphisms (SNPs) have been targeted because of their putative role on the promoter activity which might influence the TNF-α production and immunologic homeostasis.All the subjects (n: 142) included in the study were enrolled during the post monsoon period (August-December 2007-2009) i.e. JEV transmission season with informed consent. Of the total, 66 were JE patients [IgM (n: 56)/RT-PCR (n: 10) positive] with encephalitic features collected from inpatients departments of PGIMER, Chandigarh, 16 were JE IgM positive cases who presented with acute febrile illness and the rest (n: 60) were apparently healthy subjects previously exposed to JEV infection (confirmed by detection of JEV specific IgG antibody) collected from the rural areas endemic to Japanese encephalitis belonging to the neighboring rice growing states of Chandigarh. A clinical-case definition of JE was followed as per National Vector Borne Disease Control Programme, Govt. of India. Febrile illness of variable severity associated with neurological symptoms ranging from headache to meningitis or encephalitis were labeled as Acute Encephalitic Syndrome (AES). Symptoms can include headache, fever, meningeal sign, disorientation, coma, tremor, generalized paralysis, hypertonia and loss of coordination. Patients with suspected AES showing JE specific IgM antibody in the cerebro-spinal fluid (CSF) only/in paired samples of CSF and blood or JEV RNA in CSF  are labeGenomic DNA was extracted as per the manufacturer's protocol using QIAGEN DNA extraction kit. For the detection of SNPs at TNF-α promoter region, gene specific PCR was performed followed by restriction fragment length polymorphism (PCR-RFLP). The positions -863C/A, -857C/T, -308G/A, and -238G/A were amplified using the published primer sequences and amplification profiles . These Pχ2) analysis using standard statistical software with a P value of < 0.05 being statistically significant. Genotypic frequencies were examined by Hardy-Weinberg equilibrium test. EPI version 6.2 was implemented for estimation of the χ2 value, odds ratios and 95% confidence intervals for genotypic and allelic frequencies.The data was evaluated by chi-square  and control group . Again the distribution of the homozygous CC and heterozygous CA genotypes at -863 in encephalitis groups were statistically significant as compared with control group   infections. TLR-3 mediated expression of TNF-α leads to loss of integrity of the endothelial cells in the brain, possibly by mediating the passage of viral particles and virus loaded immune cells into the CNS ,23.Following JE infection, approximately 1 in 300-1,000 persons would turn out with acute encephalitis and majority abort the infection sub clinically. The present study was aimed at the host factors governing pathogenesis of JE. There is no correlation between the disease severity and the circulating JE genotypes. However, a possible correlation is observed between the severity of JE and the level of JEV specific IgM antibody in the serum and CSF .The TNF-α gene is located within the class III region of the major histocompatibility complex (MHC) between HLA-B and DR, Figure  and its The -308A allele in the TNF-α promoter  is a G/A transition and is the most studied SNP in this gene. In case of HBV infection, the presence of -308A and -863C was associated with viral clearance , whereasSingle nucleotide polymorphisms (SNPs) in the TNF-α promoter region at positions -863C/A, -857C/T, -308G/A and -238G/A with reference to the transcription start site have been analyzed based upon the TNF secretion capacity. TNF-α -308G/A has been shown to be associated with elevated TNF-α transcriptional activity. On the other hand, polymorphism at position -863C/A in the promoter region has been reported to be associated with reduced TNF-α promoter activity and lower plasma TNF levels. As per the literature search, this is the first study to identify the role of TNF-α promoter in JE infection. Our results show that subjects with -308A and -863C alleles are more vulnerable to the severe form of JE infection. Logistic regression could not be performed in this study due to limited numbers of events which happens to be one of the limitations and warrants caution with regards to the conclusion.A combined effect of prolonged exposure to high level of pro-inflammatory cytokines (TNF-α) combined with reduced release of anti-inflammatory cytokines IL-10 and IL-4 in encephalitis conditions may cause immunologic imbalance landed with poor prognosis . Host geSubjects with -308A and -863C alleles were more vulnerable to the severe form of JE infection and of the total 9 deaths, 7 had -308A and -863C allelic distribution. At the verge of entering into the era of personalized medicine, understanding the association of mutations with infectious disease is very important. This information will help the physician to become aware of a prior possible outcome of the patient and thus to follow an appropriate management protocol for the patients.The authors declare that they have no competing interests.SKP, RRK, SP, BMM and MM collectively designed the project work. SKP has done the sample collection and experimental work. SP and MM are the clinicians of this study. SKP, RRK, and BMM analyzed the data. All the authors contributed in manuscript writing. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/12/23/prepub


While co-formulation of this lipopeptide with VLPs only resulted in marginal improvements in dendritic cell (DC) uptake, its ability to concomitantly induce DC maturation at very small doses is a feature not observed using VLPs alone or in the presence of an aluminium hydroxide-based adjuvant (Alum). Dramatically improved VLP and E2-specific antibody responses were observed in VLP+E8Pam2Cys vaccinated mice where up to 3 doses of non-adjuvanted or traditionally alum-adjuvanted VLPs was required to match the antibody titres obtained with a single dose of VLPs formulated with this lipopeptide. This result also correlated with significantly higher numbers of specific antibody secreting cells that was detected in the spleens of VLP+E8Pam2Cys vaccinated mice and greater ability of sera from these mice to neutralise the binding and uptake of VLPs by Huh7 cells. Moreover, vaccination of HLA-A2 transgenic mice with this formulation also induced better VLP-specific IFN-γ-mediated responses compared to non-adjuvanted VLPs but comparable levels to that achieved when coadministered with complete freund’s adjuvant. These results suggest overall that the immunogenicity of HCV VLPs can be significantly improved by the addition of this novel adjuvant by targeting their delivery to DCs and could therefore constitute a viable vaccine strategy for the treatment of HCV.Although many studies provide strong evidence supporting the development of HCV virus-like particle (VLP)-based vaccines, the fact that heterologous viral vectors and/or multiple dosing regimes are required to induce protective immunity indicates that it is necessary to improve their immunogenicity. In this study, we have evaluated the use of an anionic self-adjuvanting lipopeptide containing the TLR2 agonist Pam Hepatitis C virus (HCV) infection affects an estimated 200 million individuals worldwide and contributes to significant morbidity and mortality rates associated with liver cirrhosis and hepatocellular carcinoma. Approximately 80% of infected individuals do not clear the virus following acute infection and will develop chronic infection that can lead to end-stage liver disease and complications. Although treatment options using a combination of pegylated interferon-α and ribavirin are available, sustained clearance of the virus is only achieved in approximately 40% of individuals infected with HCV genotype 1 and 60–70% of those who are infected with genotypes 2 or 3 Whilst the immune correlates mediating the clearance of virus are still not entirely clear or defined, there is substantial evidence demonstrating that the development of a broad multifunctional T cell response against an array of key viral proteins such as core, E1, NS3, NS4 and NS5 during acute HCV infection is associated with disease resolution + and CD8+, epitopes are packaged in VLPs (iii) VLPs lack regulatory proteins as well as genetic material that could pose a risk of reversion or mutation (iv) encouraging results have been obtained using insect cell-derived recombinant VLPs expressing HCV antigens which induce virus-specific humoral and cellular responses Virus-like particles (VLPs) possess features which make them ideal vehicles for the delivery of viral antigens to the immune system; (i) antibody epitopes are presented in the native conformation for induction of potentially neutralising antibodies (ii) multiple T cell, CD4et al have demonstrated that mammalian cell-derived recombinant envelope proteins bind to human cells with higher affinity than those produced in yeast or insect cells and appear to be antigenically and functionally similar to the viral proteins produced in an infected host cell An important consideration in the manufacture of HCV-based VLPs is the cell-type used for their manufacture. For example, it has been shown that vaccination with recombinant HCV envelope proteins expressed in mammalian cells, but not in yeast or insect cells, protect chimpanzees from primary infection by an homologous HCV isolate 2Cys) and associates electrostatically with protein antigens significantly improving their ability to induce both humoral and cell-mediated responses In this study we evaluate the immunogenicity of mammalian cell-derived VLPs containing structural proteins  of HCV genotype 1a when delivered directly to dendritic cells (DCs) using a Toll-like receptor 2 (TLR2) targeting lipopeptide. This lipopeptide contains the TLR2 agonist dipalmitoyl-S-glyceryl-cysteine (Pam5′gCCTCTAgAgCCACCATgCATCACCATCACCATCACACAAgCACgAATCCTAAACTCAAAgAAAAACC 3′) was designed to introduce an XbaI enzyme restriction site followed by a Kozak sequence, a start codon and a His(6) tag at amino terminal end of the core protein. The reverse primer of core/E1 (5′ ggCTTAAgCCCggTgACgTgggTTTCC gCgTCgAC 3′) was designed to amplify from the sequence downstream of the region corresponding to E1/E2 cleavage site (amino acids 383/384) and to introduce an Afl II restriction site at the 3′ end. Next, the E2 genome was amplified using a forward primer (5′CgACTAgTgAAACCCACgTCACCgggggAAgTgCCggCCgC 3′) that also introduced a SpeI site at the 5′ end. The reverse E2 primer (5′CgGATATCTCATCAC gCCTCCgCTTgggATATgAgTAACATCATCC 3′) was designed to introduce a double stop codon and an EcoRV restriction site at the 3′ end of the amplicon.HCV virus-like particles were constructed using a recombinant adenovirus containing encoding the HCV structural proteins  of HCV 77H, genotype 1a. Briefly, the core/E1 genes were amplified from pBRTM_HCV 1–3011 plasmid containing the genome of HCV H77 genotype 1a (a gift from Prof C Rice). The core/E1 genes were amplified from pBRTM_HCV 1–3011 plasmid containing the genome of HCV H77 genotype 1a (a gift from Prof C Rice). The forward Core/E1 primer (The core/E1 and E2 amplicons were cloned into pGEMEasy (Promega) and subsequently subcloned and ligated to produce a construct which was verified by DNA sequencing. This construct was subsequently subcloned into pAdTrack-CMV , digested with PmeI and transformed into AdEasier-1 cells by electroporation (Bio-Rad Gene Pulser) as previously described 2.High titres of recombinant adenovirions encoding the HCV proteins (rAdHCV-CE1E2) were produced in 293T cells by serial passaging and the equivalent multiplicity of infection (MOI) was determined as described previously 8) using traditional Fmoc chemistry has been described previously 8 which has an overall charge of −8. Lipidation of E8 was then carried out by removing the Mtt protective group present on the ε-amino group of the C-terminal lysine followed by acylation of the exposed ε-amino group with two serially added serine residues. The Pam2Cys lipid moiety was then coupled according to Zeng et al 8(Pam2Cys)  .2O and 0.1% TFA in acetonitrile as the limit solvent. Lipopeptides were purified if necessary. All products presented as a single major peak on analytical RP-HPLC and had the expected mass when analysed using an Agilent series 1100 ion trap mass spectrometer.Following assembly, lipopeptides were cleaved from the solid phase support and all side-chain protecting groups removed with 88% TFA, 5% phenol, 2% TIPS, 5% water for 3 hours at room temperature. Lipopeptides were analysed by reversed phase high-pressure liquid chromatography (RP-HPLC) using a Vydac C4 column (4.6×300 mm) installed in a Waters HPLC system. The chromatogram was developed at a flow rate of 1 ml/min using 0.1% TFA in H+MHC Class II+ by flow cytometry using a FACSCaliber . D1 cells (2×105) were seeded onto a petri dish in 1 ml of fresh D1 media 2 in the presence or absence of 5 µg FITC-labelled VLPs alone or with VLPs mixed with E8Pam2Cys (0.2 pmole/ml).A line of murine BALB/c-derived DCs (D1 cells) was prepared and propagated according to the method described by Chua et al Cells were harvested 24 hours later and washed with FACs wash (1% FCS/5 mM EDTA in PBS) before fixation in 1% paraformaldehyde in PBS. To examine cellular association of VLPs, cell fluorescence was analysed by flow cytometry . For examination of intracellular uptake of VLPs, extracellular fluorescence was quenched by addition of an equal volume of 0.1 M citrate buffer (pH 4.0) containing 250 µg/ml trypan blue  prior to analysis 8Pam2Cys or lipopolysaccharide (5 µg/ml) as a positive control . In some experiments, cells were incubated with VLPs alone (5 µg/ml) or in the presence of E8Pam2Cys (0.032 µg/ml). After 16 hours, cells were harvested, washed and analysed for expression of surface Class II MHC antigen.To assess the degree of DC maturation resulting from exposure to different adjuvants, cells were exposed to varying concentrations of aluminium hydroxide gel, Alhydrogel , Eb transgenic mice (HHD mice) were obtained from the Queensland Institute for Medical Research and bred in the Animal House Facility of the Department of Microbiology and Immunology at The University of Melbourne under specific pathogen free conditions. These mice do not express H-2Db but instead express the chimeric monochain of the α1 & α2 domains of HLA-A2.1 and the α3 cytoplasmic and transmembrane domains of H-2Db linked at its N-terminus to the C terminus of human β2 microglobulin All experimental procedures involving animals were approved by the University of Melbourne’s animal ethics committee under the AEC numbers 0707207 and 061061. HLA-A2k8Pam2Cys on days 0 and 14. Spleens were removed 28 days after the second dose and splenocytes restimulated in vitro at a concentration of 3×106 cells/ml in RF-10 medium consisting of RPMI 1640 medium  supplemented with 10% fetal calf serum  7.5 mM HEPES, 2 mM L-glutamine, 76 µM 2-mercaptoethanol, 150 U/ml penicillin, 150 µg/ml streptomycin, 150 µM non-essential amino acids and 10 U/ml of recombinant IL-2  at 37°C in an atmosphere of 5% CO2. Restimulation was carried out in the presence of 10 µg of VLPs or 10 µM of an irrelevant HCV-derived HLA-A2-restricted epitope (NS5B2594–2602) that is not contained in the VLP construct.Female HHD mice (3 per group) were inoculated subcutaneously on each side of the base of tail (50 µl per dose) with VLPs (30 µg) either alone, emulsified in an equal volume of complete Freund’s adjuvant (CFA) or with an equal amount of E5 cells/ml performed in polyvinylidene fluoride (PVDF) membrane-lined 96-well plates  previously coated with 5 µg/ml anti-IFN-γ capture antibody . Cells were then cultured in the presence of 3.75×105 irradiated autologous VLP-pulsed (10 µg) splenocytes for 40 hours at 37°C and 5% CO2. After washing with PBST (PBS containing 0.05% Tween 20), biotinylated anti-IFN-γ detection antibody  was added and incubated for 2 hours at room temperature in a humidified atmosphere. Plates were then washed and streptavidin-alkaline phosphatase  added and incubated for a further 2 hours. Spots representative of IFN-γ-producing cells were developed by the addition of 100 µl of 1 mg/ml 5-bromo-4-chloro-3-indolyl phosphate in 2-amino-2-methyl-1-propanol buffer  for 30 minutes. Individual spots were counted using an AID iSpot EliSpot Reader . Analysis of variance in all experiments and all p values in this study were conducted and obtained using one-way ANOVA nonparametric statistical analysis and Tukey’s post-hoc range tests performed with Prism 5 .Cells were harvested 5 days later, washed and serial dilutions commencing at 5×108Pam2Cys. An additional dose of each formulation was administered to 3 mice from each group on day 56. Sera were prepared from blood taken on days 21, 35 and 63.Female 6–8 week old BALB/c mice were inoculated subcutaneously on each side at the base of the tail (50 µl per dose) on days 0 and 21 with either VLPs (20 µg) alone, VLPs pre-incubated with an equal volume of Alhydrogel (13 mg/ml) or 60µg of Eµg/ml) in PBSN3 overnight at 4°C. Prior to coating plates with HCV VLPs, wells were pre-incubated with Galanthus nivalis lectin  in carbonate buffer  for 30 minutes at room temperature Following removal of antigen, 100 µl of BSA (10 mg/ml) in PBS was added and plates incubated for 1 hour at room temperature before washing four times with PBST . Serial dilutions of sera obtained from immunised mice were added to wells and incubated in a humidified atmosphere overnight. After washing, bound antibody was detected using horseradish peroxidase-conjugated rabbit anti-mouse IgG antibodies  in conjunction with enzyme substrate . The reaction was stopped by addition of 50 µl of 0.05 M NaF. Titers of antibody are expressed as the reciprocal of the highest dilution of serum required to achieve an optical density of 0.2.Flat bottom 96-well polyvinyl plates were coated with either purified HCV VLPs (10 µg/ml) or recombinant E2 protein  were used as detecting reagents.For the detection of specific antibody secreting cells by ELISPOT, PVDF membrane-lined 96-well plates  were coated with 100 µl of PBS containing VLPs (10 µg/ml), recombinant E2 (10 µg/ml) or anti IgG antibody (10 µg/ml) overnight at 4°C. Plates were washed 5 times with PBS and blocked for 2 hours using RPMI 1640 medium  supplemented with 20% BSA . Wells were emptied before 5×105 Huh7 cells and 200 ng of FITC-labelled VLPs were used in a total volume of 500 µl. At the end of this incubation period, cells were washed with PBS (0.1% FCS) and then fixed in BD Cytofix . Inhibition of VLP entry was determined by flow cytometry and analysed using WEASEL 2.0 software . Sera from vaccinated mice that demonstrated a decrease in specific cellular binding of 50% or more compared to sera from naïve mice were considered to contain neutralising antibodies In order to determine any inhibition of cell entry by VLPs using antibodies present in sera of vaccinated animals, Huh7 cells were first incubated with PBS (10% FCS) for 15 min at 4°C to reduce non-specific binding of antibodies subsequently added. Cells were washed twice, resuspended in PBS (0.1% FCS) and incubated with FITC-labelled VLPs at 4°C for 1 hr. Serial dilutions of sera from vaccinated or non-vaccinated mice were then added and incubated for a further 1 hour at 37°C. For each reaction, 5×108Pam2Cys also resulted in higher levels of cell fluorescence compared to untreated DCs. The percentage of fluorescenated cells in these cultures was similar to cultures that contained FITC-VLPs alone  of genotype 1a to produce particles that harbour antigenic resemblance to virions produced in an infected host cell. Because of the essential role that DCs play in the induction of both humoral and cell-mediated responses, we first examined the ability of a spleen-derived DC line (D1 cells) to take up fluorescein isothiocyanate-labelled HCV VLPs (FITC-VLPs). Flow cytometric analysis revealed that DCs incubated with FITC-VLPs exhibited higher whole cell fluorescence intensities compared to untreated DCs indicating the presence of cell-associated VLPs . ExposurPs alone .8Pam2Cys. However, a higher percentage of fluorescenated cells were detected in those cultures compared to those exposed to FITC-VLPs alone  compared to sera obtained from mice inoculated with VLPs administered in Alhydrogel (∼30%) or in saline (∼20%).To assess the neutralising activity of antibodies induced by vaccination, we first set out to investigate if VLP entry into human hepatocyte cell line Huh7 could be inhibited. Pre-incubation of FITC-labelled VLPs (VLP-FITC) with PBS or naïve serum resulted in minimal inhibition of VLP entry . However8Pam2Cys to induce a cell-meditated immune response was examined by inoculating transgenic mice expressing the MHC class I (HLA-A2) allele but not endogenous H-2Db molecules in vitro. The results  + T cell-mediated immune responses and has been shown for multiple indications 2Cys to an antigen. The use of the anionic lipopeptide E8Pam2Cys overcomes many of the technical complexities related to this process, especially the use of covalent chemistries, by making use of electrostatic association with antigen Our previous studies have shown peptide epitope and protein-based antigens can be made far more immunogenic when covalently attached to Pam8Pam2Cys compared to the use of VLPs alone or when co-administered with Alhydrogel but up to 3 doses of non-adjuvanted or traditionally adjuvanted antigen were required to match the titres obtained with a single dose using lipopeptide. Most importantly in the context of HCV the trend translates to improved E2-specific antibody responses and the use of lower doses of VLPs to achieve this while maintaining efficacy has major advantages by providing cost benefits to vaccine manufacturers.The ability of Pam2Cys to dramatically enhance the immunogenicity of HCV VLPs was demonstrated in the improved overall antibody responses that we observed. Not only are greater antibody titres induced following vaccination with VLPs formulated with EOur studies examining the interaction of VLPs with DCs indicate that while improvements in VLP uptake mediated by this lipopeptide is minimal, its ability to concomitantly induce the maturation of DCs at very small doses is a feature not observed using VLPs alone or VLPs administered in the presence of alum. Our previous work also demonstrated that association of antigen with charged lipopeptide facilitates trafficking of antigen to lymph nodes draining from the vaccination site