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1.  Parasite Burden and Severity of Malaria in Tanzanian Children 
The New England journal of medicine  2014;370(19):1799-1808.
Background
Severe Plasmodium falciparum malaria is a major cause of death in children. The contribution of the parasite burden to the pathogenesis of severe malaria has been controversial.
Methods
We documented P. falciparum infection and disease in Tanzanian children followed from birth for an average of 2 years and for as long as 4 years.
Results
Of the 882 children in our study, 102 had severe malaria, but only 3 had more than two episodes. More than half of first episodes of severe malaria occurred after a second infection. Although parasite levels were higher on average when children had severe rather than mild disease, most children (67 of 102) had high-density infection (>2500 parasites per 200 white cells) with only mild symptoms before severe malaria, after severe malaria, or both. The incidence of severe malaria decreased considerably after infancy, whereas the incidence of high-density infection was similar among all age groups. Infections before and after episodes of severe malaria were associated with similar parasite densities. Nonuse of bed nets, placental malaria at the time of a woman’s second or subsequent delivery, high-transmission season, and absence of the sickle cell trait increased severe-malaria risk and parasite density during infections.
Conclusions
Resistance to severe malaria was not acquired after one or two mild infections. Although the parasite burden was higher on average during episodes of severe malaria, a high parasite burden was often insufficient to cause severe malaria even in children who later were susceptible. The diverging rates of severe disease and high-density infection after infancy, as well as the similar parasite burdens before and after severe malaria, indicate that naturally acquired resistance to severe malaria is not explained by improved control of parasite density. (Funded by the National Institute of Allergy and Infectious Diseases and others.)
doi:10.1056/NEJMoa1303944
PMCID: PMC4091983  PMID: 24806160
2.  Co-infection of Long-Term Carriers of Plasmodium falciparum with Schistosoma haematobium Enhances Protection from Febrile Malaria: A Prospective Cohort Study in Mali 
Background
Malaria and schistosomiasis often overlap in tropical and subtropical countries and impose tremendous disease burdens; however, the extent to which schistosomiasis modifies the risk of febrile malaria remains unclear.
Methods
We evaluated the effect of baseline S. haematobium mono-infection, baseline P. falciparum mono-infection, and co-infection with both parasites on the risk of febrile malaria in a prospective cohort study of 616 children and adults living in Kalifabougou, Mali. Individuals with S. haematobium were treated with praziquantel within 6 weeks of enrollment. Malaria episodes were detected by weekly physical examination and self-referral for 7 months. The primary outcome was time to first or only malaria episode defined as fever (≥37.5°C) and parasitemia (≥2500 asexual parasites/µl). Secondary definitions of malaria using different parasite densities were also explored.
Results
After adjusting for age, anemia status, sickle cell trait, distance from home to river, residence within a cluster of high S. haematobium transmission, and housing type, baseline P. falciparum mono-infection (n = 254) and co-infection (n = 39) were significantly associated with protection from febrile malaria by Cox regression (hazard ratios 0.71 and 0.44; P = 0.01 and 0.02; reference group: uninfected at baseline). Baseline S. haematobium mono-infection (n = 23) did not associate with malaria protection in the adjusted analysis, but this may be due to lack of statistical power. Anemia significantly interacted with co-infection (P = 0.009), and the malaria-protective effect of co-infection was strongest in non-anemic individuals. Co-infection was an independent negative predictor of lower parasite density at the first febrile malaria episode.
Conclusions
Co-infection with S. haematobium and P. falciparum is significantly associated with reduced risk of febrile malaria in long-term asymptomatic carriers of P. falciparum. Future studies are needed to determine whether co-infection induces immunomodulatory mechanisms that protect against febrile malaria or whether genetic, behavioral, or environmental factors not accounted for here explain these findings.
Author Summary
The parasitic diseases malaria and schistosomiasis are tremendous public health burdens, each affecting over 200 million people worldwide with substantial geographic overlap in sub-Saharan Africa. Understanding how schistosomiasis influences the human immune response to Plasmodium, the agent of malaria, can be important for developing effective malaria vaccines. Past studies have tried to determine if infection with Schistosoma haematobium, which causes urinary schistosomiasis, affects the number of febrile attacks from malaria caused by Plasmodium falciparum in communities where the diseases overlap, but the findings have been inconsistent. Here, we examined 616 healthy people from a village in Mali for symptomless infections with S. haematobium and treated those with infections. We then followed them over a single malaria-transmission season of 7 months during which we diagnosed and treated all febrile malaria attacks. After the season, we examined archived blood collected at enrollment to look for occult P. falciparum infection. The study revealed that people who were infected with both parasites at the beginning of the season were better protected from the malaria attacks than those who were uninfected or infected with either parasite alone. Further studies are needed to confirm these findings and to determine the biological basis for this phenomenon.
doi:10.1371/journal.pntd.0003154
PMCID: PMC4161351  PMID: 25210876
3.  Quantile Regression for Recurrent Gap Time Data 
Biometrics  2013;69(2):375-385.
Summary
Evaluating covariate effects on gap times between successive recurrent events is of interest in many medical and public health studies. While most existing methods for recurrent gap time analysis focus on modeling the hazard function of gap times, a direct interpretation of the covariate effects on the gap times is not available through these methods. In this article, we consider quantile regression that can provide direct assessment of covariate effects on the quantiles of the gap time distribution. Following the spirit of the weighted risk-set method by Luo and Huang (2011, Statistics in Medicine 30, 301–311), we extend the martingale-based estimating equation method considered by Peng and Huang (2008, Journal of the American Statistical Association 103, 637–649) for univariate survival data to analyze recurrent gap time data. The proposed estimation procedure can be easily implemented in existing software for univariate censored quantile regression. Uniform consistency and weak convergence of the proposed estimators are established. Monte Carlo studies demonstrate the effectiveness of the proposed method. An application to data from the Danish Psychiatric Central Register is presented to illustrate the methods developed in this article.
doi:10.1111/biom.12010
PMCID: PMC4123128  PMID: 23489055
Clustered survival data; Data perturbation; Gap times; Quantile regression; Recurrent events; Within-cluster resampling
4.  B-Cell Activating Factor (BAFF) is elevated in Chronic Granulomatous Disease 
Clinical immunology (Orlando, Fla.)  2013;148(2):258-264.
Chronic Granulomatous Disease (CGD) is an inherited defect in superoxide production leading to life-threatening infections, granulomas, and, possibly, abnormal immunoglobulin concentrations. We investigated whether factors controlling antibody production, such as B-cell activating factor (BAFF), were altered in CGD. CGD subjects had significantly increased mean (2.3-fold, p<0.0001) plasma concentrations of BAFF compared to healthy donors. Patients on IFN-γ treatment had significantly higher BAFF concentrations compared with CGD patients not taking IFN-γ (1.6-fold, p<0.005). Leukocytes from CGD subjects produced normal amounts of BAFF in response to IFN-γ or G-CSF in vitro. Expression of BAFF-R and TACI were significantly reduced on CGD B cells. Elevated BAFF in CGD correlated with CRP (R=0.44), ESR (R=0.49), and IgM (R=0.47) and increased rapidly in healthy subjects following intravenous endotoxin administration. These findings suggest that elevated BAFF in CGD subjects and healthy donors is a consequence of acute and chronic inflammation.
doi:10.1016/j.clim.2013.05.007
PMCID: PMC3774275  PMID: 23773925
Chronic Granulomatous Disease; B-cell activating factor; inflammation
5.  Clinical characteristics and outcomes of influenza and other influenza-like illnesses in Mexico City 
Summary
Background
Influenza-like illnesses (ILI) are estimated to cause millions of deaths annually. Despite this disease burden, the etiologic causes of ILI are poorly described for many geographical regions.
Methods
Beginning in April 2010, we conducted an observational cohort study at five hospitals in Mexico City, enrolling subjects who met the criteria for ILI. Evaluations were conducted at enrollment and on day 28, with the collection of clinical data and a nasopharyngeal swab (or nasal aspirate in children). Swabs were tested by multiplex PCR for 15 viral pathogens and real-time PCR for influenza.
Results
During the first year, 1065 subjects were enrolled in this study, 55% of whom were hospitalized; 24% of all subjects were children. One or more pathogens were detected by PCR in 64% of subjects, most commonly rhinovirus (25% of all isolates) and influenza (24% of isolates). Six percent of subjects died, and of those, 54% had no pathogen identified. Rhinovirus was the most common pathogen among those who died, although it did not have the highest case fatality rate.
Conclusions
Multiple respiratory viruses beyond influenza are associated with significant morbidity and mortality among adults and children in Mexico City. Detection of these agents could be useful for the adjustment of antibiotic treatment in severe cases.
doi:10.1016/j.ijid.2013.01.006
PMCID: PMC3655081  PMID: 23416208
Respiratory viral pathogens; Epidemiology; Hospital burden of disease; Influenza; Respiratory syncytial virus; Rhinovirus; Coronavirus
6.  An Intensive Longitudinal Cohort Study of Malian Children and Adults Reveals No Evidence of Acquired Immunity to Plasmodium falciparum Infection 
To investigate the risk of Plasmodium falciparum infection in a cohort of naturally exposed individuals, we assessed the time to first P. falciparum infection by polymerase chain reaction analysis of dried blood spots and found no age-related differences in infection risk.
Background In experimental models of human and mouse malaria, sterilizing liver stage immunity that blocks progression of Plasmodium infection to the symptomatic blood stage can be readily demonstrated. However, it remains unclear whether individuals in malaria-endemic areas acquire such immunity.
Methods In Mali, 251 healthy children and adults aged 4–25 years who were free of blood-stage Plasmodium infection by polymerase chain reaction (PCR) were enrolled in a longitudinal study just prior to an intense 6-month malaria season. Subsequent clinical malaria episodes were detected by weekly active surveillance and self-referral. Asymptomatic P. falciparum infections were detected by blood-smear microscopy and PCR analysis of dried blood spots that had been collected every 2 weeks for 7 months.
Results As expected, the risk of clinical malaria decreased with increasing age (log-rank test, P = .0038). However, analysis of PCR data showed no age-related differences in P. falciparum infection risk (log-rank test, P = .37).
Conclusions Despite years of exposure to intense P. falciparum transmission, there is no evidence of acquired, sterile immunity to P. falciparum infection in this population, even as clinical immunity to blood-stage malaria is clearly acquired. Understanding why repeated P. falciparum infections do not induce sterile protection may lead to insights for developing vaccines that target the liver stage in malaria-endemic populations.
doi:10.1093/cid/cit174
PMCID: PMC3669526  PMID: 23487390
preerythrocytic immunity; endemic population; Plasmodium falciparum; infection risk; malaria
7.  Molecular Signatures of Hemagglutinin Stem-Directed Heterosubtypic Human Neutralizing Antibodies against Influenza A Viruses 
PLoS Pathogens  2014;10(5):e1004103.
Recent studies have shown high usage of the IGHV1-69 germline immunoglobulin gene for influenza hemagglutinin stem-directed broadly-neutralizing antibodies (HV1-69-sBnAbs). Here we show that a major structural solution for these HV1-69-sBnAbs is achieved through a critical triad comprising two CDR-H2 loop anchor residues (a hydrophobic residue at position 53 (Ile or Met) and Phe54), and CDR-H3-Tyr at positions 98±1; together with distinctive V-segment CDR amino acid substitutions that occur in positions sparse in AID/polymerase-η recognition motifs. A semi-synthetic IGHV1-69 phage-display library screen designed to investigate AID/polη restrictions resulted in the isolation of HV1-69-sBnAbs that featured a distinctive Ile52Ser mutation in the CDR-H2 loop, a universal CDR-H3 Tyr at position 98 or 99, and required as little as two additional substitutions for heterosubtypic neutralizing activity. The functional importance of the Ile52Ser mutation was confirmed by mutagenesis and by BCR studies. Structural modeling suggests that substitution of a small amino acid at position 52 (or 52a) facilitates the insertion of CDR-H2 Phe54 and CDR-H3-Tyr into adjacent pockets on the stem. These results support the concept that activation and expansion of a defined subset of IGHV1-69-encoded B cells to produce potent HV1-69-sBnAbs does not necessarily require a heavily diversified V-segment acquired through recycling/reentry into the germinal center; rather, the incorporation of distinctive amino acid substitutions by Phase 2 long-patch error-prone repair of AID-induced mutations or by random non-AID SHM events may be sufficient. We propose that these routes of B cell maturation should be further investigated and exploited as a pathway for HV1-69-sBnAb elicitation by vaccination.
Author Summary
The quest for universal influenza vaccine has gained wide interest with the discovery of human neutralizing antibodies that are able to variably cross neutralize and protect against different influenza strains, subtypes, groups and lineages. These antibodies, which bind to a highly conserved epitope in the hemagglutinin stem, are often encoded by rearranged IGHV1-69 germline genes that alone make contact with HA and prevent virus entry and emergence of escape mutants. Our study was undertaken to gain an understanding of what structural requirements enable a rearranged IGHV1-69 Ab to become a potent cross-neutralizing antibody. We found that in addition to a critical amino acid triad consisting of a pair of anchor residues in CDR-H2 and a properly positioned CDR-H3 Tyr, distinctive V-segment substitutions that arise in positions that are distinct from phase I AID somatic hypermutation (SHM) hotspot motifs are often required. As few as two V-segment SHM can fulfill this role which appears to facilitate the optimal binding of CDR-H2 Phe54 and CHR-H3-Tyr into adjacent hydrophobic pockets in the HA stem. These studies provide new information on the SHM requirements for IGHV1-69-encoded B cells to produce HV1-69-sBnAbs and suggest that there may exist alternative routes to their elicitation by vaccination.
doi:10.1371/journal.ppat.1004103
PMCID: PMC4006906  PMID: 24788925
8.  Joint Modeling and Estimation for Recurrent Event Processes and Failure Time Data 
Recurrent event data are commonly encountered in longitudinal follow-up studies related to biomedical science, econometrics, reliability, and demography. In many studies, recurrent events serve as important measurements for evaluating disease progression, health deterioration, or insurance risk. When analyzing recurrent event data, an independent censoring condition is typically required for the construction of statistical methods. In some situations, however, the terminating time for observing recurrent events could be correlated with the recurrent event process, thus violating the assumption of independent censoring. In this article, we consider joint modeling of a recurrent event process and a failure time in which a common subject-specific latent variable is used to model the association between the intensity of the recurrent event process and the hazard of the failure time. The proposed joint model is flexible in that no parametric assumptions on the distributions of censoring times and latent variables are made, and under the model, informative censoring is allowed for observing both the recurrent events and failure times. We propose a “borrow-strength estimation procedure” by first estimating the value of the latent variable from recurrent event data, then using the estimated value in the failure time model. Some interesting implications and trajectories of the proposed model are presented. Properties of the regression parameter estimates and the estimated baseline cumulative hazard functions are also studied.
PMCID: PMC3780991  PMID: 24068850
Borrow-strength method; Frailty; Informative censoring; Joint model; Nonstationary Poisson process
9.  Kernel Estimation of Rate Function for Recurrent Event Data 
Recurrent event data are largely characterized by the rate function but smoothing techniques for estimating the rate function have never been rigorously developed or studied in statistical literature. This paper considers the moment and least squares methods for estimating the rate function from recurrent event data. With an independent censoring assumption on the recurrent event process, we study statistical properties of the proposed estimators and propose bootstrap procedures for the bandwidth selection and for the approximation of confidence intervals in the estimation of the occurrence rate function. It is identified that the moment method without resmoothing via a smaller bandwidth will produce a curve with nicks occurring at the censoring times, whereas there is no such problem with the least squares method. Furthermore, the asymptotic variance of the least squares estimator is shown to be smaller under regularity conditions. However, in the implementation of the bootstrap procedures, the moment method is computationally more efficient than the least squares method because the former approach uses condensed bootstrap data. The performance of the proposed procedures is studied through Monte Carlo simulations and an epidemiological example on intravenous drug users.
doi:10.1111/j.1467-9469.2005.00416.x
PMCID: PMC3779323  PMID: 24062598
bootstrap; independent censoring; intensity function; kernel estimator; Poisson process; rate function; recurrent events
10.  Composite Partial Likelihood Estimation Under Length-Biased Sampling, With Application to a Prevalent Cohort Study of Dementia 
The Canadian Study of Health and Aging (CSHA) employed a prevalent cohort design to study survival after onset of dementia, where patients with dementia were sampled and the onset time of dementia was determined retrospectively. The prevalent cohort sampling scheme favors individuals who survive longer. Thus, the observed survival times are subject to length bias. In recent years, there has been a rising interest in developing estimation procedures for prevalent cohort survival data that not only account for length bias but also actually exploit the incidence distribution of the disease to improve efficiency. This article considers semiparametric estimation of the Cox model for the time from dementia onset to death under a stationarity assumption with respect to the disease incidence. Under the stationarity condition, the semiparametric maximum likelihood estimation is expected to be fully efficient yet difficult to perform for statistical practitioners, as the likelihood depends on the baseline hazard function in a complicated way. Moreover, the asymptotic properties of the semiparametric maximum likelihood estimator are not well-studied. Motivated by the composite likelihood method (Besag 1974), we develop a composite partial likelihood method that retains the simplicity of the popular partial likelihood estimator and can be easily performed using standard statistical software. When applied to the CSHA data, the proposed method estimates a significant difference in survival between the vascular dementia group and the possible Alzheimer’s disease group, while the partial likelihood method for left-truncated and right-censored data yields a greater standard error and a 95% confidence interval covering 0, thus highlighting the practical value of employing a more efficient methodology. To check the assumption of stable disease for the CSHA data, we also present new graphical and numerical tests in the article. The R code used to obtain the maximum composite partial likelihood estimator for the CSHA data is available in the online Supplementary Material, posted on the journal web site.
doi:10.1080/01621459.2012.682544
PMCID: PMC3758493  PMID: 24000265
Backward and forward recurrence time; Cross-sectional sampling; Random truncation; Renewal processes
11.  Analysing panel count data with informative observation times 
Biometrika  2006;93(4):763-775.
Summary
In this paper, we study panel count data with informative observation times. We assume nonparametric and semiparametric proportional rate models for the underlying event process, where the form of the baseline rate function is left unspecified and a subject-specific frailty variable inflates or deflates the rate function multiplicatively. The proposed models allow the event processes and observation times to be correlated through their connections with the unobserved frailty; moreover, the distributions of both the frailty variable and observation times are considered as nuisance parameters. The baseline rate function and the regression parameters are estimated by maximising a conditional likelihood function of observed event counts and solving estimation equations. Large-sample properties of the proposed estimators are studied. Numerical studies demonstrate that the proposed estimation procedures perform well for moderate sample sizes. An application to a bladder tumour study is presented.
doi:10.1093/biomet/93.4.763
PMCID: PMC3666563  PMID: 23729818
Dependent censoring; Frailty; Poisson process; Rate function; Serial events
12.  Empirical Likelihood-Based Estimation of the Treatment Effect in a Pretest–Posttest Study 
The pretest–posttest study design is commonly used in medical and social science research to assess the effect of a treatment or an intervention. Recently, interest has been rising in developing inference procedures that improve efficiency while relaxing assumptions used in the pretest–posttest data analysis, especially when the posttest measurement might be missing. In this article we propose a semiparametric estimation procedure based on empirical likelihood (EL) that incorporates the common baseline covariate information to improve efficiency. The proposed method also yields an asymptotically unbiased estimate of the response distribution. Thus functions of the response distribution, such as the median, can be estimated straightforwardly, and the EL method can provide a more appealing estimate of the treatment effect for skewed data. We show that, compared with existing methods, the proposed EL estimator has appealing theoretical properties, especially when the working model for the underlying relationship between the pretest and posttest measurements is misspecified. A series of simulation studies demonstrates that the EL-based estimator outperforms its competitors when the working model is misspecified and the data are missing at random. We illustrate the methods by analyzing data from an AIDS clinical trial (ACTG 175).
doi:10.1198/016214508000000625
PMCID: PMC3666595  PMID: 23729942
Auxiliary information; Biased sampling; Causal inference; Observational study; Survey sampling
13.  The Impact of Genetic Susceptibility to Systemic Lupus Erythematosus on Placental Malaria in Mice 
PLoS ONE  2013;8(5):e62820.
Severe malaria, including cerebral malaria (CM) and placental malaria (PM), have been recognized to have many of the features of uncontrolled inflammation. We recently showed that in mice genetic susceptibility to the lethal inflammatory autoimmune disease, systemic lupus erythematosus (SLE), conferred resistance to CM. Protection appeared to be mediated by immune mechanisms that allowed SLE-prone mice, prior to the onset of overt SLE symptoms, to better control their inflammatory response to Plasmodium infection. Here we extend these findings to ask does SLE susceptibility have 1) a cost to reproductive fitness and/or 2) an effect on PM in mice? The rates of conception for WT and SLE susceptible (SLEs) mice were similar as were the number and viability of fetuses in pregnant WT and SLEs mice indicating that SLE susceptibility does not have a reproductive cost. We found that Plasmodium chabaudi AS (Pc) infection disrupted early stages of pregnancy before the placenta was completely formed resulting in massive decidual necrosis 8 days after conception. Pc-infected pregnant SLEs mice had significantly more fetuses (∼1.8 fold) but SLE did not significantly affect fetal viability in infected animals. This was despite the fact that Pc-infected pregnant SLEs mice had more severe symptoms of malaria as compared to Pc-infected pregnant WT mice. Thus, although SLE susceptibility was not protective in PM in mice it also did not have a negative impact on reproductive fitness.
doi:10.1371/journal.pone.0062820
PMCID: PMC3651086  PMID: 23675429
14.  A maximum pseudo-profile likelihood estimator for the Cox model under length-biased sampling 
Biometrika  2012;99(1):199-210.
This paper considers semiparametric estimation of the Cox proportional hazards model for right-censored and length-biased data arising from prevalent sampling. To exploit the special structure of length-biased sampling, we propose a maximum pseudo-profile likelihood estimator, which can handle time-dependent covariates and is consistent under covariate-dependent censoring. Simulation studies show that the proposed estimator is more efficient than its competitors. A data analysis illustrates the methods and theory.
doi:10.1093/biomet/asr072
PMCID: PMC3667656  PMID: 23843659
Approximate likelihood; Cross-sectional sampling; Product-limit estimator; Random truncation; Screening trials
15.  Safety and Immunogenicity of Multiple and Higher Doses of an Inactivated Influenza A/H5N1 Vaccine 
The Journal of infectious diseases  2009;200(4):501-509.
Background
H5N1 avian influenza represents an episodic zoonotic disease with potential to cause a pandemic, and resistance is of considerable concern. We sought to generate high titer H5N1 antibodies in healthy volunteers for the purpose of developing hyperimmune IVIG.
Methods
We conducted a dose escalating unblinded clinical trial involving 75 subjects between the ages of 18 and 59 years. Three cohorts of twenty-five subjects were enrolled sequentially receiving 90, 120, or 180 mcg of H5N1 A/Vietnam/1203/04 vaccine for four doses 28 days apart.
Results
No significant dose related increases in the geometric mean titers (GMTs) of serum HAI were observed when comparing 90, 120, and 180 mcg. When analyzed together to determine the effect of additional vaccinations, HAI GMT after first, second, third and fourth vaccinations was 1:15.7, 1:22.2, 1:36.0, and 1:32.0, respectively (first vs baseline, P<0.0001; second vs first, P=0.02; third vs second, P<0.0001). The MN GMT after first, second, third, and fourth vaccinations was 1:17.5, 1:33.1, 1:55.7, and 1:68.4 respectively (all P<0.001).
Conclusion
Our study suggests that a third dose and fourth dose of the H5N1 A/Vietnam/1203/04 vaccine may result in a higher HA and MN GMT. There was no benefit to increasing the dose of the vaccine.
doi:10.1086/599992
PMCID: PMC3417327  PMID: 19569973
avian influenza; vaccination; passive immunotherapy; IVIG
16.  A model checking method for the proportional hazards model with recurrent gap time data 
Biostatistics (Oxford, England)  2010;12(3):535-547.
Recurrent events are the natural outcome in many medical and epidemiology studies. To assess covariate effects on the gaps between consecutive recurrent events, the Cox proportional hazards model is frequently employed in data analysis. The validity of statistical inference, however, depends on the appropriateness of the Cox model. In this paper, we propose a class of graphical techniques and formal tests for checking the Cox model with recurrent gap time data. The building block of our model checking method is an averaged martingale-like process, based on which a class of multiparameter stochastic processes is proposed. This maneuver is very general and can be used to assess different aspects of model fit. Numerical simulations are conducted to examine finite-sample performance, and the proposed model checking techniques are illustrated with data from the Danish Psychiatric Central Register.
doi:10.1093/biostatistics/kxq071
PMCID: PMC3138070  PMID: 21138876
Correlated failure times; Induced-dependent censoring; Kaplan–Meier estimator; Renewal processes
17.  Wide Prevalence of Heterosubtypic Broadly Neutralizing Human Anti-Influenza A Antibodies 
Sera from H5N1 vaccinees and IVIG were analyzed for crossreactive Abs against HA of influenza A viruses. Data quantitatively shows the presence of two populations of heterosubtypic neutralizing Abs against either Group 1 only or Group 1 + 2 viruses.
(See the editorial commentary by Donis and Cox, on pages 1010–1012.)
Background. Lack of life-long immunity against influenza viruses represents a major global health care problem with profound medical and economic consequences. A greater understanding of the broad-spectrum “heterosubtypic” neutralizing human antibody (BnAb) response to influenza should bring us closer toward a universal influenza vaccine.
Methods. Serum samples obtained from 77 volunteers in an H5N1 vaccine study were analyzed for cross-reactive antibodies (Abs) against both subtype hemagglutinins (HAs) and a highly conserved pocket on the HA stem of Group 1 viruses. Cross-reactive Abs in commercial intravenous immunoglobulin were affinity purified using H5-coupled beads followed by step-wise monoclonal antibody competition or acid elution. Enzyme-linked immunosorbent assays were used to quantify cross-binding, and neutralization activity was determined with HA-pseudotyped viruses.
Results. Prevaccination serum samples have detectable levels of heterosubtypic HA binding activity to both Group 1 and 2 influenza A viruses, including subtypes H5 and H7, respectively, to which study subjects had not been vaccinated. Two different populations of Broadly neutralizing Abs (BnAbs) were purified from intravenous immunoglobulin by H5 beads: ∼0.01% of total immunoglobulin G can bind to HAs from both Group 1 and 2 and neutralize H1N1 and H5N1 viruses; ∼0.001% is F10-like Abs directed against the HA stem pocket on Group 1 viruses.
Conclusion. These data—to our knowledge, for the first time—quantitatively show the presence, albeit at low levels, of two populations of heterosubtypic BnAbs against influenza A in human serum. These observations warrant further investigation to determine their origin, host polymorphism(s) that may affect their expression levels and how to boost these BnAb responses by vaccination to reach sustainable protective levels.
doi:10.1093/cid/cir121
PMCID: PMC3070035  PMID: 21460314
18.  Nonparametric estimation for length-biased and right-censored data 
Biometrika  2011;98(1):177-186.
Summary
This paper considers survival data arising from length-biased sampling, where the survival times are left truncated by uniformly distributed random truncation times. We propose a nonparametric estimator that incorporates the information about the length-biased sampling scheme. The new estimator retains the simplicity of the truncation product-limit estimator with a closed-form expression, and has a small efficiency loss compared with the nonparametric maximum likelihood estimator, which requires an iterative algorithm. Moreover, the asymptotic variance of the proposed estimator has a closed form, and a variance estimator is easily obtained by plug-in methods. Numerical simulation studies with practical sample sizes are conducted to compare the performance of the proposed method with its competitors. A data analysis of the Canadian Study of Health and Aging is conducted to illustrate the methods and theory.
doi:10.1093/biomet/asq069
PMCID: PMC3412605  PMID: 23049126
Backward and forward recurrence time; Cross-sectional sampling; Partial likelihood; Random truncation; Renewal process
19.  Development of Functional and Molecular Correlates of Vaccine-Induced Protection for a Model Intracellular Pathogen, F. tularensis LVS 
PLoS Pathogens  2012;8(1):e1002494.
In contrast with common human infections for which vaccine efficacy can be evaluated directly in field studies, alternative strategies are needed to evaluate efficacy for slowly developing or sporadic diseases like tularemia. For diseases such as these caused by intracellular bacteria, serological measures of antibodies are generally not predictive. Here, we used vaccines varying in efficacy to explore development of clinically useful correlates of protection for intracellular bacteria, using Francisella tularensis as an experimental model. F. tularensis is an intracellular bacterium classified as Category A bioterrorism agent which causes tularemia. The primary vaccine candidate in the U.S., called Live Vaccine Strain (LVS), has been the subject of ongoing clinical studies; however, safety and efficacy are not well established, and LVS is not licensed by the U.S. FDA. Using a mouse model, we compared the in vivo efficacy of a panel of qualitatively different Francisella vaccine candidates, the in vitro functional activity of immune lymphocytes derived from vaccinated mice, and relative gene expression in immune lymphocytes. Integrated analyses showed that the hierarchy of protection in vivo engendered by qualitatively different vaccines was reflected by the degree of lymphocytes' in vitro activity in controlling the intramacrophage growth of Francisella. Thus, this assay may be a functional correlate. Further, the strength of protection was significantly related to the degree of up-regulation of expression of a panel of genes in cells recovered from the assay. These included IFN-γ, IL-6, IL-12Rβ2, T-bet, SOCS-1, and IL-18bp. Taken together, the results indicate that an in vitro assay that detects control of bacterial growth, and/or a selected panel of mediators, may ultimately be developed to predict the outcome of vaccine efficacy and to complement clinical trials. The overall approach may be applicable to intracellular pathogens in general.
Author Summary
Diseases such as tuberculosis (caused by Mycobacterium tuberculosis) or tularemia (caused by Francisella tularensis) result from infections by microbes that live within cells of a person's body. New vaccines are being developed against such intracellular pathogens, but some will be difficult to test, because disease takes a long time to develop (e.g., tuberculosis) or because outbreaks are unpredictable (e.g., tularemia). Usually such infections are controlled by activities of T cells. However, there are no accepted measures of T cell function that reliably predict vaccine-induced protection. We studied two new ways to do so. We used a group of vaccine candidates against tularemia that stimulated good, fair, or poor protection of mice against Francisella challenge. We then measured whether Francisella–immune cells from vaccinated mice controlled the growth of bacteria inside cells, and/or whether the expression of immune genes in Francisella–immune cells was increased. We found that the degree of protection was matched by the degree of the cells' function in controlling intramacrophage bacterial growth. Further, the degree was predicted by relative amounts of gene expression for several immune mediators. Thus the two new options explored here may help predict protection, without waiting for the onset of disease.
doi:10.1371/journal.ppat.1002494
PMCID: PMC3262015  PMID: 22275868
20.  Interferon-α Produces Significant Decreases in HIV Load 
A randomized, controlled clinical trial was started in the pre-HAART era to compare the efficacy of zidovudine (AZT) and interferon-alpha (IFN-α) either alone or in combination to reduce HIV viremia, maintain CD4+ cell count, and decrease time to AIDS progression and death. The purpose of the current study was to compare the effects of AZT and IFN on HIV load using modern technology. One hundred and eighty patients with CD4+ counts above 500 cells/mm3 were randomized to receive AZT alone, IFN-α alone, or AZT and IFN-α in combination. CD4+ cell count and HIV load at baseline and at the last follow-up visit were compared, and time to AIDS or death was calculated by treatment group. At a mean follow-up of 45 weeks, the mean change in log HIV RNA was −0.06 for the AZT alone group, −0.47 for the AZT plus IFN-α group (P = 0.01 versus AZT group), and −0.35 for the IFN-α alone group (P = 0.02 versus AZT group). There was no significant difference among groups in change in total CD4+ count or in time to AIDS or death. Since treatment with IFN-α produces significant decreases in HIV load, additional studies of IFN-α as part of a combination regimen are warranted.
doi:10.1089/jir.2009.0090
PMCID: PMC2964361  PMID: 20235638
21.  The TLR9 agonist CpG fails to enhance the acquisition of Plasmodium falciparum-specific memory B cells in semi-immune adults in Mali 
Vaccine  2009;27(52):7299-7303.
Antibodies play a key role in controlling blood stage malaria infections, and an effective blood stage malaria vaccine will likely require that it induce vaccine-specific memory B cells (MBCs). Our previous studies showed that the addition of the TLR9 agonist CpG to Plasmodium falciparum protein subunit vaccines greatly increased their efficacy in inducing MBCs in nonimmune U.S. volunteers. Here we show that in contrast the same CpG-containing malaria vaccine did not enhance the acquisition of MBCs in semi-immune adults living in Mali. Understanding the molecular basis of this apparent refractoriness to TLR9 agonist will be of significant interest in vaccine design.
doi:10.1016/j.vaccine.2009.08.023
PMCID: PMC2788035  PMID: 19712767
Memory B cells; Plasmodium falciparum; Malaria; Toll-like receptor 9
22.  The TLR9 ligand CpG promotes the acquisition of Plasmodium falciparum-specific memory B cells in malaria-naïve individuals 
Despite the central role of memory B cells (MBC) in protective immune responses, little is understood about how they are acquired in naïve individuals in response to antigen exposure, and how this process is influenced by concurrent activation of the innate immune system’s Toll-like receptors (TLR). In this longitudinal study of malaria-naïve individuals, we examined the MBC response to two candidate malaria vaccines administered with or without CpG, a TLR9 ligand. We show that the acquisition of MBC is a dynamic process in which the vaccine-specific MBC pool rapidly expands and then contracts, and that CpG enhances the kinetics, magnitude, and longevity of this response. We observed that the percentage of vaccine-specific MBC present at the time of re-immunization predicts vaccine-specific Ab levels 14 days later; and that at steady state, there is a positive correlation between vaccine-specific MBC and Ab levels. An examination of the total circulating MBC and plasma cell (PC) pools also suggests that MBC differentiate into PC through polyclonal activation, independent of antigen specificity. These results provide important insights into the human MBC response which can inform the development of vaccines against malaria and other pathogens that disrupt immunological memory.
doi:10.4049/jimmunol.0803596
PMCID: PMC2910392  PMID: 19234231
Human; B cells; Memory; Parasitic-Protozoan; Vaccination
23.  The Plasmodium falciparum-Specific Human Memory B Cell Compartment Expands Gradually with Repeated Malaria Infections 
PLoS Pathogens  2010;6(5):e1000912.
Immunity to Plasmodium falciparum (Pf) malaria is only acquired after years of repeated infections and wanes rapidly without ongoing parasite exposure. Antibodies are central to malaria immunity, yet little is known about the B-cell biology that underlies the inefficient acquisition of Pf-specific humoral immunity. This year-long prospective study in Mali of 185 individuals aged 2 to 25 years shows that Pf-specific memory B-cells and antibodies are acquired gradually in a stepwise fashion over years of repeated Pf exposure. Both Pf-specific memory B cells and antibody titers increased after acute malaria and then, after six months of decreased Pf exposure, contracted to a point slightly higher than pre-infection levels. This inefficient, stepwise expansion of both the Pf-specific memory B-cell and long-lived antibody compartments depends on Pf exposure rather than age, based on the comparator response to tetanus vaccination that was efficient and stable. These observations lend new insights into the cellular basis of the delayed acquisition of malaria immunity.
Author Summary
Plasmodium falciparum (Pf) is a mosquito-borne parasite that causes over 500 million cases of malaria annually, one million of which result in death, primarily among African children. The development of an effective malaria vaccine would be a critical step toward the control and eventual elimination of this disease. To date, most licensed vaccines are for pathogens that induce long-lived protective antibodies after a single infection. In contrast, immunity to malaria is only acquired after repeated infections. Antibodies play a key role in protection from malaria, yet several studies indicate that antibodies against some Pf proteins are generated inefficiently and lost rapidly. The cells that are responsible for the maintenance of antibodies over the human lifespan are memory B-cells and long-lived plasma cells. To determine how these cells are generated and maintained in response to Pf infection, we conducted a year-long study in an area of Mali that experiences a six-month malaria season. We found memory B-cells and long-lived antibodies specific for the parasite were generated in a gradual, step-wise fashion over years despite intense Pf exposure. This contrasts sharply with the efficient response to tetanus vaccination in the same population. This study lends new insights into the delayed acquisition of malaria immunity. Future studies of the cellular and molecular basis of these observations could open the door to strategies for the development of a highly effective malaria vaccine.
doi:10.1371/journal.ppat.1000912
PMCID: PMC2873912  PMID: 20502681
24.  Antifungal Activities of Natural and Synthetic Iron Chelators Alone and in Combination with Azole and Polyene Antibiotics against Aspergillus fumigatus▿  
Antifungal effects of iron chelators (lactoferrin, deferoxamine, deferiprone, and ciclopirox) were tested alone and in combination with antifungal drugs against Aspergillus fumigatus B5233 conidia. Lactoferrin, ciclopirox, and deferiprone inhibited whereas deferoxamine enhanced fungal growth. Antifungal synergy against conidia was observed for combinations of ketoconazole with ciclopirox or deferiprone, lactoferrin with amphotericin B, and fluconazole with deferiprone. Iron chelation alone or combined with antifungal drugs may be useful for prevention and treatment of mycosis.
doi:10.1128/AAC.01547-08
PMCID: PMC2687209  PMID: 19307370
25.  In Vitro Growth-Inhibitory Activity and Malaria Risk in a Cohort Study in Mali▿ † 
Infection and Immunity  2009;78(2):737-745.
Immunity to the asexual blood stage of Plasmodium falciparum is complex and likely involves several effector mechanisms. Antibodies are thought to play a critical role in malaria immunity, and a corresponding in vitro correlate of antibody-mediated immunity has long been sought to facilitate malaria vaccine development. The growth inhibition assay (GIA) measures the capacity of antibodies to limit red blood cell (RBC) invasion and/or growth of P. falciparum in vitro. In humans, naturally acquired and vaccine-induced P. falciparum-specific antibodies have growth-inhibitory activity, but it is unclear if growth-inhibitory activity correlates with protection from clinical disease. In a longitudinal study in Mali, purified IgGs, obtained from plasmas collected before the malaria season from 220 individuals aged 2 to 10 and 18 to 25 years, were assayed for growth-inhibitory activity. Malaria episodes were recorded by passive surveillance over the subsequent 6-month malaria season. Logistic regression showed that greater age (odds ratio [OR], 0.78; 95% confidence interval [95% CI], 0.63 to 0.95; P = 0.02) and growth-inhibitory activity (OR, 0.50; 95% CI, 0.30 to 0.85; P = 0.01) were significantly associated with decreased malaria risk in children. A growth-inhibitory activity level of 40% was determined to be the optimal cutoff for discriminating malaria-immune and susceptible individuals in this cohort, with a sensitivity of 97.0%, but a low specificity of 24.3%, which limited the assay's ability to accurately predict protective immunity and to serve as an in vitro correlate of antibody-mediated immunity. These data suggest that antibodies which block merozoite invasion of RBC and/or inhibit the intra-RBC growth of the parasite contribute to but are not sufficient for the acquisition of malaria immunity.
doi:10.1128/IAI.00960-09
PMCID: PMC2812204  PMID: 19917712

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