Highly active antiretroviral therapy (HAART) for the treatment of individuals infected by HIV-1 is limited by high costs, drug resistance, and drug-related toxicities. This has led researchers to investigate new treatment options, including ways to boost immune responses to better control HIV. One such approach has been termed supervised treatment interruption (STI)—in which HAART is intermittently stopped once viral load has been reduced to a low level, in order to boost natural immunity by brief exposure to virus. The goal is to allow for the eventual discontinuation of drug treatment.Nature in 2000, suggested that this approach worked in persons treated in the earliest stages of acute HIV infection. HIV-1 viral loads in newly infected patients remained suppressed for a median of six months after therapy had been stopped. However, a follow up paper, published this month in PLoS Medicine by the same research group, shows that the viral load rebounded in eight of the 14 patients by one year.Preliminary evidence, published by Bruce Walker and his colleagues from Harvard Medical School in “The findings are very straightforward and very important,” comments Danny Douek from the Vaccine Research Center, National Institutes of Health, United States, who was not involved in the study. “In almost every case, virus rebounded and no clinical benefit from the interruption could be determined.”Walker's team first considered the possibility of STI in 1997 after they demonstrated that HAART given to patients recently infected with HIV could protect T helper cells, which are normally destroyed in the earliest stages of infection. They hypothesized that early treatment of acute HIV-1 infection with HAART might boost the immune response, allowing it to control the HIV-1 infection without the need for continuous therapy. “We did not know at that time whether the T helper cells would be functional,” explains Walker. “The only way to tell this was to stop medications and see if the immune response could control the virus.”Nature paper. “The key finding was that we were able to get at least transient control of virus in all eight persons studied, and in five of eight the viral load was less than 500 copies (very low!) at the time of publication,” explains Walker. However, at that point they did not know how long the protective effects would last.To test this hypothesis the researchers did an open-label trial of STIs; they published data from six months follow-up in the The first evidence that protection was not complete came two years later when Walker's team reported a case of superinfection; one of the patients in the original experiment was infected with a second strain of HIV, even though the first virus was still well controlled. “This paper was important because it indicated that the amount of immunity might be enough for the person's own virus, but might not protect against closely related viruses circulating in the population,” says Walker.PLoS Medicine study adds more concern since it shows that although most persons can indeed transiently control their own virus, they do so for only a limited amount of time. “We expanded the study to 14 persons, and now have about five years of follow-up on some of the patients,” says Walker. “Although we were able to use early treatment and structured treatment interruption to boost immunity and have 11 of 14 patients control their virus, most of the persons ultimately ‘broke through,’ meaning that they had a recurrence of viremia.” At the present time the researchers do not know what causes the loss of viral control.The Walker and colleagues conclude that treatment interruptions should probably be avoided outside the setting of controlled clinical trials, whereas Douek goes a step further: “The study shows that even early short-term treatment and structured treatment interruptions, using current strategies, impart only transient benefit and are unlikely to serve as a reasonable therapeutic option in the future.”


The strikingly young age of new variant Creutzfeldt-Jacob disease (vCJD) cases remains unexplained. Age dependent susceptibility to infection has been put forward, but differential dietary exposure to contaminated food products in the UK population according to age and sex during the bovine spongiform encephalopathy (BSE) epidemic may provide a simpler explanation.Using recently published estimates of dietary exposure in mathematical models of the epidemiology of the new variant Creutzfeldt Jacob disease (vCJD), we examine whether the age characteristics of vCJD cases may be reproduced.The susceptibility/exposure risk function has likely peaked in adolescents and was followed by a sharp decrease with age, evocative of the profile of exposure to bovine material consumption according to age. However, assuming that the risk of contamination was proportional to exposure, with no age dependent susceptibility, the model failed to reproduce the observed age characteristics of the vCJD cases: The predicted cumulated proportion of cases over 40 years was 48%, in strong disagreement with the observed 10%. Incorporating age dependent susceptibility led to a cumulated proportion of cases over 40 years old of 12%.This analysis provides evidence that differential dietary exposure alone fails to explain the pattern of age in vCJD cases. Decreasing age related susceptibility is required to reproduce the characteristics of the age distribution of vCJD cases. Owing to the lack of data concerning age dependent dietary exposure to bovine material in food in the UK, mathematical models used in the study of new variant Creutzfeldt-Jacob disease (vCJD) have postulated an age risk function for contamination where the influence of age dependent dietary exposure to contaminated material could not be separated from intrinsic age related susceptibility -3. For eBy June 2003, 139 cases of vCJD had been reported to the vCJD surveillance unit in the UK, as compared to the 79 cases used in our original paper . This ofWe therefore set out to estimate the age risk function with a versatile description based on step functions, and to investigate whether dietary exposure alone could explain the age distribution of cases.We obtained the age, sex and date of onset for the 137 vCJD cases reported to the UK vCJD unit as of June 2003, all of which had onset before October 2002. Delays in reporting may reach 18 months  and bias downward the incidence curve. Therefore, we included in our analysis only the 129 cases with onset before November 2001, consisting in 71 men and 58 women. Mortality by age and sex for the UK was obtained from the Office for National Statistics [λ(a,t), was assumed to depend on date t and age a in a multiplicative manner, with λ(a,t) = f(a) g(t), where  parallels the entry of infected cows in the human food chain estimated from back-calculation results in the UK [r corresponds to the impact of the specified risk material ban in 1989; f(a) is a function varying with age only (see following paragraph). Occurrence of cases of vCJD of sex i  is then modelled by a Poisson process in the (age, time) plane, with intensity , so that the expected number of onsets of sex i in the period [t,t+dt) and age [a,a+da) is πi(a,t) da dt [h is the distribution of the incubation time, Si(a,t) is the probability of survival at time t for individuals of sex i aged a calculated from census information, and βι is the intensity of a homogeneous Poisson process. The incubation period distribution h was taken to be lognormal, as the choice of a particular shape is not critical for the estimates [The model extends our previously described method . The insn the UK  and r cot) da dt ,7. In thstimates . This di, where the first sum is on all observed cases where si is the sex of individual i, and the second is over both sexes (s) and requires integration of the intensity over the domain (in time and age) D = [0,100] × [1 Jan 1980,31 Nov 2001]. Custom FORTRAN code, using SLATEC and TOMS library for numerical routines, was used in this step. In all instances, our model allowed estimation of the mean and standard deviation of the incubation period, the shape of f(a), and β.Estimates of the parameters were obtained by numerical maximization of the log-likelihood, defined as (a,t) over the period [1/1/1980, 1/11/2001] for consecutive age class of width 10 years. The χ2 distance was computed to measure agreement between the observed and the model calculated age distributions, but no formal test was carried out.The model predicted distribution of age for cases with onset before November 2001 was determined by integrating To allow a versatile shape in the age risk function, we chose a non parametric description based on step functions rather than a mathematical function with few parameters. so that the shape of f(a) was unconstrained for a<25 and decreased exponentially after age 25. We then estimated fi, i = 1,...,6 and α at maximum likelihood. With this particular choice, it is possible to visually check the progression in risk with age during infancy (f1, f2, f3), the presence of a peak in risk in teenagers (f2,f3,f4), and whether the drop in risk after 20 is strong enough to be exponential (f4, f5, f6).We wrote e(a) as the product of mean consumption by the corresponding percentage of consumers in age class a, and wrote f(a) = f0 e(a), where f0 was estimated at maximum likelihood.To investigate whether dietary exposure to meat was sufficient to explain the young age of vCJD cases, we first determined the exposure to meat according to age and sex from UK data, using consumption of carcass meat . In thes and likewise estimated fi from the data.Finally, to estimate the influence of age standardised on dietary exposure, we wrote Susceptibility & Diet" and "Susceptibility | Diet", we normalised the fi by the largest of the values in the graphical presentation of results, yielding risks relative to the highest risk age class.In model ", where θ stands for a vector of k parameters,  for the estimates, and i(θ) for the Fisher information matrix, has a limiting chi-squared distribution with k degrees of freedom. We therefore determined 95% confidence intervals by finding the values of parameters so that Q(θ) was less than the 95th quantile of the corresponding chi-squared distribution.Maximum likelihood estimators have a limiting normal distribution around the true parameters, with limiting variance/covariance matrix given by the inverse of the Fisher information , therefoSusceptibility & Diet" is presented in Figure χ2 = 2.45; Figure The estimated age risk function in model "Diet Alone", while the average incubation period was estimated at 12.1 (CI 95% [10.2, 14.2]) years with a standard deviation of 2.4 (CI95% [1.2, 4.1])years, close to that of the first model, the predicted age distribution of the cases was at odds with the observed distribution (χ2 = 58.4), as apparent in Figure Diet Alone" assumption, when the observed percentage was only 10%; it was 12% with the "Susceptibility & Diet" model.The shape of the estimated age risk function in Figure Susceptibility | Diet" led to a profile for the fi as shown in Figure Susceptibility & Diet" model, although with an earlier maximum susceptibility. With this model, the average incubation period established at 12.6 years (CI 95% [10.5, 14.7]) with standard error 1.8 years (CI95% [1.2, 3.5]), and the predicted age distribution of cases showed agreement similar to that of the "Susceptibility & Diet" model (χ2 = 2.36).When estimating the residual influence of age, once exposure has been taken into account, model "Incorporating differential dietary exposure to BSE infected products according to age and sex, in a flexible age risk function for vCJD contamination, we found that exposure alone could not explain the young age of vCJD cases seen in the UK. Decreasing age related susceptibility had to be assumed to reproduce the characteristics of the age distribution of these cases.In all instances, the estimates for the epidemiological characteristics of vCJD were in line with those previously reported from comparable number of cases ,9,10, anSusceptibility & Diet", it appears that a previous assumption of a constant age risk in children and adolescents [From the model "lescents  has likelescents , althougSusceptibility | Diet" model. This finding confirms that obtained in a recent analysis based on scenario analysis, where it was found that exponential decrease in susceptibility in the oldest cohorts was desirable [However, even after adjustment for dietary exposure, susceptibility remains rapidly decreasing in adults, as found in the "esirable . This isn = 129).Three hypotheses have been put forward to explain the young age of the vCJD cases: age dependent incubation period, age dependent exposure, and age dependent susceptibility. Age dependent incubation period has recently been revived by Cooper et al., because scenarios including longer incubation periods in old cohorts provided a better fit of the data. Howeverχ2 test, P = 0.49) although the proportion of males is 55%. Our analysis shows that, while the estimated age risk function for infection with vCJD exhibits a peak in the young that was also found in dietary exposure to potentially BSE infected products, differential dietary exposure alone does not account for the observed pattern in the age of the vCJD cases. Indeed, the relative exposure does not decrease rapidly enough with age to reduce the risk in older adults. Therefore, an additional effect of age is required to fully account for the age of the vCJD cases. Once exposure is taken into account, this effect appears to be peaking in children less than 10 years old, and decreasing afterwards.The potential role of differential dietary exposure was proposed early in the epidemic , becauseContamination through BSE infected food is today the most plausible explanation for the occurrence of vCJD; this is the rationale for correcting the risk of infection by the level of dietary exposure. However, this explanation remains today hypothetical, because even if epidemiology and biochemistry favour a link between BSE and vCJD, this is not regarded as ultimately conclusive . In thisThe reasons for an increased susceptibility in teenagers compared to young children and adults are today speculative. In many infectious diseases, a decrease in susceptibility with age is mediated through the immunological responses to repeated exposure since birth. However, for vCJD, most cohorts in the population have had the same duration of exposure to BSE infected material. Due to the age range considered, biological processes involved in the maturation of the immune system; in response to hormonal changes may be incriminated. For example, changes in the permeability of the intestinal barrier occurs with age with decreasing Peyer's patches . FurthervCJD: variant Creutzfeldt-Jacob DiseaseBSE : Bovine Spongiform EncephalopathyNone declared.PYB designed the study, analysed the results and drafted the manuscript. JYC participated in the design and writing. AJV participated in the design and writing. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


In a recent publication it was claimed that cultured mammalian cells, in contrast to yeasts, maintain a constant size distribution in the population without a size checkpoint. This inference may be challengeable.(1) It is argued that "weak" size control implies the existence of a checkpoint, and unfortunately the technique used by Conlon and Raff might obscure such a weak mechanism. (2) Previous investigations of size control in yeasts have shown that individual cell data, rather than means and variances of cell populations, are prerequisites for reliable interpretation. (3) No experimental data so far obtained suggest that in any cell culture a linear growth pattern in cell mass can maintain size homeostasis on its own without size control. (4) Studies on fission yeast mutants indicate that the molecular mechanisms of size control vary with genetic background, implying that no single mechanism is likely to apply to any cell type, including cultured mammalian cells, under all conditions.The claim that cultured mammalian cells maintain size homeostasis without a checkpoint needs to be re-evaluated by measurements on individual cells. This isThere is a long history of size measurements on fission yeast and the involvement of size in the control of the cell cycle, see e.g. -5, but oOne of the two cases cited by Conlon and Raff as evidence for the lack of a size checkpoint is the slow change in size when cells are stimulated by fresh medium . The reawithout a size checkpoint. It is plausible that this might happen because of the general need for co-ordination between the cytoplasmic and chromosome cycles, but we are not convinced that linear growth on its own would meet this requirement. In the case of fission yeast, there is evidence that linear growth without a size checkpoint cannot maintain homeostasis in the culture (see below).Another problem in  is the icdc mutants was approximately exponential over a longer period after this type of block. This result comes from an early paper and depends on measurements of absolute amounts of protein and RNA/ml [cdc2-33, the rate of protein synthesis reaches a plateau after 4 h [cdc13 [Related observations from fission yeast show that normal size control is abolished in the type of block experiment used in ; howeverd RNA/ml . There ifter 4 h . Also, oh [cdc13 . Becauseh [cdc13 , which mwee mutants , but this size control is a strong one, albeit weaker in diploids [wee1-50 cdc25Δ [There are other points about the fission yeast experiments that are relevant to size control, which is almost certainly affected by genetic background. The main rate change point (an effect of gene-dosage, see above) is shifted in position in diploids . There a0 cdc25Δ . It seemcdc mutants of fission yeast, and size control is lost for some cycles (but not forever), as indicated by the lack of negative correlation between length extension and birth length [wee1-50 rum1Δ, the cells lose viability after a few generations [Another point is that the normal rules of the cycle can be broken temporarily, but not for long. For example, mammalian cells can certainly grow without entering S phase ; and theh length ,3. Howeverations  despite erations . Accordierations , cell siTo return to mammalian cells: our view is that the best way forward is to try to devise ways of following individual cells through the cycle. It is easy for us to say this, since we have had the great advantage of working with regular shaped yeast cells that stay still and grow steadily on agar. The mammalian cells situation is far more difficult, but there is reasonable hope that it can be solved by methods of tracking individual cells such as fibroblasts by a semi-automatic method and measuring their dry mass by interferometry . The mai


Cells receive and send signals across the plasma membrane using the integrin family of receptors. What is it about their structure that can mediate their function? It goes without saying that the cellular plasma membrane effectively creates a barrier between the inside (intracellular area) and outside (extracellular area) of the cell it defines. In order for the cell to sense and respond to its environment (including other cells and the supporting structures that comprise the extracellular matrix [ECM]) and for the environment to influence cell function (including cell growth and movement), bidirectional signaling across the plasma membrane has to be mediated by receptors and other structures. About two decades ago, it became widely appreciated that many of the cell surface receptors that mediate cell–cell and cell–ECM interactions were structurally and functionally related, and the term “integrins” was coined to reflect the capacity of members of this family to integrate the extracellular and intracellular environment . IntegriThe integrin family comprises 20 or more members that are found in many animal species, ranging from sponges to mammals . They coMutational studies provided the initial hints that disruption of the non-covalent clasp between α and β cytoplasmic tails is clearly the event within the structure of the integrin that initiates inside-out signaling. Point mutations in the α and β cytoplasmic tails that are near the membrane or deletion of either region result in constitutive activation of the receptor . Mutatinvβ3 (the nomenclature identifies the particular α and β subunits) was determined  interacts with itself—called homotypic oligomerization of the transmembrane domains  Li et a. Ligand Despite the molecular level of our understanding of integrin activation, a number of key questions remain unresolved. Although we know that the membrane-proximal clasp on the integrin cytoplasmic face controls the integrin activation, the distal side of either the α or β cytoplasmic tails may also play a role in integrin activation, since other mutations indicate that the C-terminal membrane distal region is important in regulating integrin activation via a mechanism that is yet unknown. Thus, the picture for the cytoplasmic face-controlled inside-out activation may be substantially more complicated than specified in Upon the inside-out activation, integrins bind to specific extracellular matrix proteins. However, for the integrins to grip tightly to the extracellular matrix to mediate cell adhesion and migration, the integrin cytoplasmic domains must be anchored to the cytoskeleton . This is


The immune system has a remarkable capacity for fending off infectious diseases, and it has become clear that these same defenses can recognize and destroy cancer cells. In fact, they do so on an ongoing basis, and cancer develops only when immune surveillance breaks down. Many patients with established tumors also mount an immune response against some antigens that are specific to, or enriched in, the tumor. This response, however, is rarely effective against the disease.The idea of enlisting the immune system to fight cancer has been around for a long time, and has led to the development of various cancer vaccines designed to alert the immune system to the presence of a tumor and to induce a response that, selectively and potently, will eliminate tumor cells. Vaccines include whole tumor extracts or specific proteins and peptides that are selectively expressed or enriched in tumors, by themselves or with a variety of adjuvants.There have been some spectacular successes, in particular with immune therapy to malignant melanoma, a tumor type that seems naturally to be more immunogenic than others. However, even in melanoma, success is usually restricted to a fraction of the patients, with no obvious explanation of why the strategy works for a particular patient and fails in most others. The emphasis has consequently shifted from clinical outcomes to monitoring a patient's immune response. What type of response is necessary and sufficient to eliminate tumor cells is still unclear, but the hope is that understanding the immune response in patients that show clinical benefit will answer that question.Peter Lee and colleagues used state-of-the art technology to dissect the endogenous immune response to vaccination with heteroclitic melanoma peptides, i.e., melanoma-associated peptides that have been engineered to elicit a stronger immune response. They focused on cytotoxic T lymphocytes (CTLs), and compared CTL clones from four melanoma patients who had vaccine-induced T cell responses and two melanoma patients with spontaneous anti-tumor T cell responses. The researchers analyzed several hundred CTL clones  for T cell receptor variable chain beta expression, recognition efficiency, and ability to lyse target melanoma cells. Most T cells isolated from vaccinated patients were poor at tumor cell lysis compared with T cells from endogenous responses to cancer.The authors suggest that the high doses of peptides administered in vaccinations and the increased binding capacity of heteroclitic peptides to major histocompatibility complex molecules—the very quality that makes them more immunogenic—induce many T cells with low recognition efficiency for the native peptides they encounter on the tumor cells. Their findings also bring into question the ability to deduce the recognition efficiency and tumor reactivity of T cell responses from ELISPOT and tetramer staining assays—the two standard measures of T cell responses to vaccines—which has implications for rational vaccine design in general.


In these regions, substitution rates are suppressed, which results in an overall substitution process that is not Poissonian. However, the simulation method of Bastolla et al. is representative only for cases in which the product of mutation rate μ and population size Ne is small. How the substitution process behaves when μNe is large is not known.A frequent observation in molecular evolution is that amino-acid substitution rates show an index of dispersion (that is, ratio of variance to mean) substantially larger than one. This observation has been termed the overdispersed molecular clock. On the basis of in silico protein evolution as a function of mutation rate and population size. I find that the index of dispersion decays with increasing μNe, and approaches 1 for large μNe . This observation can be explained with the selective pressure for mutational robustness, which is effective when μNe is large. This pressure keeps the population out of low-neutrality traps, and thus steadies the ticking of the molecular clock.Here, I study the behavior of the molecular clock in μNe, and a mostly Poissonian one for large μNe. The former is relevant for the majority of organisms in the plant and animal kingdom, and the latter may be relevant for RNA viruses.The molecular clock in neutral protein evolution can fall into two distinct regimes, a strongly overdispersed one for small  Kimura has argued that the majority of nucleotide substitutions that accumulate in genes over time are selectively neutral, and go to fixation purely by chance . One majin silico, and found significant overdispersion in all cases they considered.An explanation for fluctuations in neutral mutation rate was recently proposed by Bastolla et al. -16. Diffμ and population size Ne is small . Since population size and mutation rate can be substantial in some species (most notably in RNA viruses), it is justified to ask how general this result is for arbitrary values of μNe. It is known that large populations and populations evolving under high mutation pressure experience a strong selective pressure to avoid regions of low neutrality, an effect that has been termed "evolution of mutational robustness" [However, the simulations that Bastolla et al. carried out were limited to cases in which the product of mutation rate ustness" -20. In eμ and population size Ne. I have found that the accumulation of non-synonymous mutations is substantially overdispersed for small μNe, in agreement with the results of Bastolla et al., but approaches a Poisson process when μNe ≫ 1. The accumulation of synonymous substitutions is always Poissonian, regardless of the value of μNe.For the present paper, I have studied the behavior of the substitution process under neutral protein evolution as a function of mutation rate L = 75. I determined the fitness of a DNA sequence by translating it into the corresponding amino-acid sequence, and determining its native fold within the framework of a lattice-protein model. (A sequence would have fitness 1 if it folded into a pre-determined target structure, and fitness 0 otherwise.) I used a simple model of maximally compact proteins on a 5 × 5 lattice. This protein-folding model is much simpler than the ones used by Bastolla et al. [I carried out simulations with DNA sequences of length a et al. ,14, but a et al. -23. The R substantially above 1) for non-synonymous substitutions, but not for synonymous substitutions. The finding for synonymous mutations is not surprising, because changes in the protein's neutrality do not affect the probability with which a synonymous mutation is neutral . Neutral evolution could produce overdispersion in the synonymous substitutions only if the number of synonymous sites in the sequence were undergoing significant fluctuations. While these fluctuations do occur, they are apparently not large enough to affect the index of dispersion.First, I have found that my model produces overdispersion  Figure μNe between approximately 10 and 100, in agreement with Ref. [μτ.For non-synonymous substitutions, μNe ≳ 10 . Therefocumulate . Thus, iith Ref. , and thaR increases with μτ. This dependency becomes clearer in Fig. R as a function of μτ for fixed μNe. The figure shows substantial increase in R with increasing μτ for small to moderate μNe. However, even for μNe well above 50, there is still a slight increase in R with μτ. Therefore, my results do not settle the question of whether the substitution process becomes truly Poissonian for sufficiently large μNe, or whether it just approaches a Poisson process but always remains slightly overdispersed. To settle this question, one would have to carry out simulations with much larger τ and Ne. Unfortunately, the protein folding model I use is still too computationally intensive to permit such simulations with current computational resources.Figure μNe has a substantial effect on the index of dispersion under neutral evolution. The substitution process is strongly overdispersed for small μNe, but approaches a Poisson process as μNe grows large. Therefore, the next question is which of the two regimes has more biological relevance. As discussed by Cutler [My results show that the size of the product y Cutler , the bigDrosophila. Keightley and Eyre-Walker estimated the per-nucleotide substitution rate in Drosophila to be u = 2.2 × 10-9 [Drosophila is 1770 bp long [μn = 3.0 × 10-6. With an effective population size of approximately 3 × 105 [Drosophila lies well within the parameter region in which we expect overdispersion to be caused by neutral evolution. For other higher organisms, in particular mammals, which tend to have comparatively small population sizes, we can expect that the product μnNe falls into the same parameter region. On the other hand, for microorganisms, which can have very large population sizes, mutational robustness may play a role in their evolution. In particular, RNA viruses have genomic mutation rates on the order of one [Ne is much smaller than the number of virus particles in infected individuals (which can exceed 1012), and is more closely related to the number of infected individuals. For HIV-1, Ne has been estimated to be approximately 102 for subtype A, and 105 for subtype B [To assess the likelihood that fluctuations in neutrality contribute to the overdispersed molecular clock, we have to know the mutation rate and population size for the species of interest. It is notoriously difficult to obtain accurate data for these parameters, and only a few species have been studied in depth. One of the best data sets available is probably the one for 2 × 10-9 . If we a bp long , and tha 3 × 105 , we get r of one ,27 and tubtype B .Drosophila and other organisms. However, overdispersion has been observed in synonymous substitutions as well. For example, Zeng et al. [R significantly above one for synonymous, but not for non-synonymous substitutions in Drosophila. For mammals, some studies found R significantly above one for both synonymous and non-synonymous substitutions [The preceeding paragraph shows that neutral evolution of proteins is probably one source of overdispersed non-synonymous substitutions in g et al.  found anitutions , while oitutions . TherefoμNe results in a substitution process with little overdispersion. However, I have not yet given an explanation for how overdispersion is reduced in populations with large μNe. There are two elements: First, selection for mutational robustness reduces the fraction of sequences with low neutrality, and increases the fraction of sequences with high neutrality, thus making the population more homogeneous and reducing the overall range of neutralities [μNe, and will therefore have a reduced probability to end up on the line of descent. While this selective disadvantage is often small, it can nevertheless determine the evolutionary fate of a sequence in a large population. The larger the population, the more sensitive it becomes to small fitness differences, so that in a very large population a sequence with only a moderate reduction in neutrality will have a small probability to end up on the line of descent. ]/ [1 - exp(2sNe)], where s is the selective disadvantage of the deleterious mutation [μNe, as long as Ne is large in comparison to s.Throughout this paper, I have considered only neutral or lethal mutations. It is a reasonable question to ask if and how deleterious mutations would change my results. The answer is that they probably have only a minor impact, and the less so the larger mutation . TherefoThe present study supports the following conclusions:• Neutral drift of proteins can lead to an overdispersed substitution process for non-synonymous mutations, but not for synonymous mutations.μNe ≈ 10, and extends to values well above 100.• The amount of overdispersion in the non-synonymous substitution process depends strongly on the product of mutation rate and population size. As this product increases, the substitution process becomes more and more Poissonian. The transition region starts at Drosophila, the product of mutation rate and population size is close to one, which is well below the parameter region in which the substitution process turns Poissonian.• It is not clear whether there are any species that have a sufficiently large population size and mutation rate to prevent overdispersion through neutral drift. In by Goldstein and coworkers [n = 25 residues that fold into a maximally compact structure on a two-dimensional grid of 5 × 5 lattice points. There are 1081 distinct possible conformations in this model, and the partition function can be evaluated exactly by summing over the contact energies of all distinct conformations.I implemented a version of the 5 × 5 lattice protein model put forward oworkers -23,32. Ii isThe contact energy of a conformation  is the contact energy between amino acids  at location j and  at location k in the sequence, and  is 1 if the two amino acids are in contact in conformation k, and 0 otherwise. The partition function iswhere f if the contact energy for that conformation is lower than the contact energies of all other formations, Ef <Ei for all i ≠ f, and if the free energy of folding, which is defined aswhere the sum runs over all 1081 conformations. A sequence folds into conformation Gcut. Throughout this study, I used kT = 0.6 and ΔGcut = 0. The contact energies  where taken from Table VI in Ref. [is smaller than some cutoff Δ in Ref. .Ne. The fitness of a sequence was 1 if the DNA sequence translated into a peptide sequence that could fold into a chosen target structure, and had a free energy of folding smaller than Gcut. Otherwise, the fitness of the sequence was 0. All sequences had length L = 75. In each successive generation, sequences with fitness 1 were randomly chosen to reproduce, until the new generation had Ne members. At reproduction, the sequences were mutated, with an average of μ base pair substitutions per sequence. I let each population evolve for several thousand generations, and kept track of the full genealogic information of all sequences in the population. In order to measure the molecular clock of fixed mutations only, I studied the pattern of base substitutions in a window of τ generations along the line of descent backwards in time, starting from the most recent common ancestor of the final population.I simulated the evolution of populations of DNA sequences in discrete, non-overlapping generations. Population size is denoted by Ne (10, 33, 100, 330, 1000, 3300), μ (0.0075, 0.075, 0.75), and τ (500, 1000). For each set of parameters, I carried out 500 replicates (each with a different, randomly chosen target structure), to obtain a distribution for the number of synonymous and non-synonymous substitutions Sd and Nd. Since there was some variation in the number of synonymous and non-synonymous sites across different target structures (on the order of approximately ± 5% variation from the mean), I then applied a correction factor to Sd and Nd to bring them into comparable units: I calculated the corrected number of synonymous substitutions  as  Here, S is the mean number of synonymous sites for the given replicate, and (S) is the average of S over all 500 replicates. Likewise, I calculated  . Similar correction factors have been used in sequence analysis [I varied the parameters To obtain an estimate for mean and standard error of the index of dispersion, I subdivided the 500 results into 10 blocks of 50 each, and calculated mean and variance of the number of substitutions for each block. The ratio of variance to mean for a given set of substitutions (synonymous or non-synonymous) in a block is the index of dispersion for this data set. I then calculated mean and standard error for the index of dispersion from the individual results of the 10 blocks.The total CPU time needed to carry out all simulations was several months on a small cluster of Pentium II 500 MHz machines.S and N and the number of synonymous and non-synonymous substitutions Sd and Nd according to the method proposed by Nei and Gojobori [si of a codon i is the fraction of possible substitutions to that codon that leave the residue unchanged. The number of non-synonymous sites ni for the same codon is ni = 3 - si. For the complete sequence, S and N are calculated as  and  where i runs over all codons in the sequence. The number of synonymous or non-synonymous substitutions si d,or ni d,between two codons is the average number of such substitutions, where the average is taken over all paths that lead from one codon to the other. The total number of synonymous or non-synonymous substitutions between two sequences is the sum over all individual constributions,  and  .I calculated the number of synonymous and non-synonymous sites Gojobori . In shorTo calculate the number of synonymous or non-synonymous substitutions along the line of descent, I simply summed up all synonymous or non-synonymous substitutions that occurred from generation to generation. Because the full evolutionary history was known, a correction for multiple mutations such as the Jukes-Cantor correction  was not COW carried out all aspects of this study.


Viruses are obligate intracellular parasites that rely upon the host cell for different steps in their life cycles. The characterization of cellular genes required for virus infection and/or cell killing will be essential for understanding viral life cycles, and may provide cellular targets for new antiviral therapies.5 independently disrupted genes, of which 111 contained mutations in previously characterized genes and functionally anonymous transcription units. Collectively, the genes associated with reovirus resistance differed from genes targeted by random gene entrapment in that known mutational hot spots were under represented, and a number of mutations appeared to cluster around specific cellular processes, including: IGF-II expression/signalling, vesicular transport/cytoskeletal trafficking and apoptosis. Notably, several of the genes have been directly implicated in the replication of reovirus and other viruses at different steps in the viral lifecycle.Candidate genes required for lytic reovirus infection were identified by tagged sequence mutagenesis, a process that permits rapid identification of genes disrupted by gene entrapment. One hundred fifty-one reovirus resistant clones were selected from cell libraries containing 2 × 10Tagged sequence mutagenesis provides a rapid, genome-wide strategy to identify candidate cellular genes required for virus infection. The candidate genes provide a starting point for mechanistic studies of cellular processes that participate in the virus lifecycle and may provide targets for novel anti-viral therapies. Cellular genes are likely to participate in all phases of viral life cycles including attachment to cellular receptors, internalization, disassembly, translation of mRNA, assembly and egress from the cells . The susGene traps are efficient mutagens as assessed by studies in mice of mutations induced originally in embryonic stem cells. In somatic cells, the approach assumes that loss-of-function mutations induced by gene entrapment may confer reovirus resistance as a result of gene dosage effects (e.g. haploinsufficiency), pre-existing heterozygosity or loss of heterozygosity. Following infection with the U3NeoSV1 retrovirus gene trap shuttle vector, libraries of mutagenized rat intestinal epithelial (RIE)-1 cell clones were isolated in which each clone contained a single gene disrupted by provirus integration . The entCtcf mutation, a transcriptional repressor of insulin growth factor II (IGF-II), was one of 4 mutations associated with reovirus resistance that affected IGF-II expression and/or signalling. Subsequent experiments demonstrated that enforced IGF-II expression is sufficient to confer high levels of reovirus resistance [Reovirus-resistant clones were selected at higher frequencies from entrapment libraries than from non-mutagenized cells, suggesting that reovirus-resistant phenotypes were induced by gene trap mutagenesis. However in any genetic screen, clones with the selected phenotype may arise from spontaneous mutations, and consequently, additional experiments are required to demonstrate that individual genes disrupted by gene entrapment actually contribute to the reovirus-resistant phenotype. For example, a mutation in 4 independent gene trap events, were isolated following infection with the U3NeoSV1 gene trap retrovirus. U3NeoSV1 contains coding sequences for a neomycin resistance gene in the U3 region of the viral long terminal repeat (LTR). Selection for neomycin resistance generates clones with proviruses inserted within actively transcribed genes. Cells pooled from each entrapment library were separately infected with Type 1 reovirus at a multiplicity of infection of 35, and reovirus-resistant clones were selected in serum-free media to suppress the emergence of persistently infected (PI) cells (4). A total of 151 reovirus-resistant clones were isolated – approximately 1 mutant per 103 gene trap clones or 1 mutant per 107 reovirus infected cells. For comparison, the frequency of recovering resistant clones from RIE-1 cells not mutagenized by gene entrapment was less than 10-8. This suggests that reovirus-resistant phenotypes were induced by gene trap mutagenesis.Twenty libraries of mutagenized RIE-1 cells, each representing approximately 10Reovirus-resistant cells selected in serum-free media did not express viral antigens Figure  and did The U3NeoSV1 gene trap vector contains a plasmid origin of replication and ampicillin resistance gene; thus, regions of genomic DNA adjacent to the targeting vector were readily cloned by plasmid rescue and sequenced . The flaClr)-f and -C, Dnaja1-/Aprataxin+ (Aprx), GATA binding protein 4 (Gata4), Bcl2 like-1 (Bcl2l1); and chromosome 10 open reading frame 3 (Chr10orf3) and myoferlin, fer-1 like protein 3 (Fer1l3), S100a6 , and two functionally anonymous cDNAs were independently mutated in separate cell libraries (Supplementary Table [see The Supplementary Table see  lists geable see ). The prNeo gene to produce enzymatically active Neo fusion proteins. As a consequence, mutagenesis by U3NeoSV1 is actually quite biased, such that of 400 mutations characterized in ES cells, one-third involved genes disrupted multiple times, including Pecam1 which was targeted 9 times [Madh7 and Gas5, each represented once among the reovirus-resistant clones, were disrupted by U3NeoSV1 in ES cells. Both genes are commonly targeted by other retroviral gene trap vectors and thus probably represent hot spots for gene entrapment [While the presence of multiple, independent mutations in specific genes provides indirect evidence for their involvement in the reovirus lifecycle, the genes could also represent hot spots for gene entrapment. The U3NeoSV1 vector preferentially targets genes with 5' exons that can splice in-frame with a cryptic splice site in the  9 times . Howevertrapment .Igf2r) transports cathepsins to the lysosome [+-ATPase (Atp6v0c) [4Cl is a weak base that interferes with the function of two of the tagged genes, the Igf2r and the Atp6v0c, and blocks the disassembly of reovirus and several other viruses that enter cells via the endocytic pathway. Moreover, specific inhibitors of the vacuolar H+-ATPase gene product have been used to block the infectivity of reovirus and influenza A virus [Igf2r, Prss11, a protease associated with insulin binding protein 5, and Ctcf a transcriptional repressor of IGF-II] are predicted to affect IGF-II expression and/or signalling. Cells containing the Ctcf mutation were subsequently found to express elevated levels IGF-II, while enforced IGF-II expression was sufficient to confer high levels of reovirus resistance. The resistance was caused, at least in part, by a block in virus disassembly [The genes associated with reovirus resistance can be grouped according to their presumed role in virus entry, disassembly, translation, and maturation. Reovirus enters the host via an endocytic pathway that requires acidification and proteolysis to remove the viral outer capsid. The presumptive roles of several candidate genes would be anticipated to affect virus replication by interfering with virus disassembly. For example the mannose-6-phosphate receptor/insulin-growth factor-2 receptor (lysosome  and acidAtp6v0c) . NH4Cl i A virus ,14. Fourassembly . By infeS100a6/S100a1) – proteins that may bind to each other [Cald1, Kif13b, Mapt, Mkln1, Stmn1, Tm9sf4, and Tpm1). Annexin-II associates with cytomegalovirus virions and anti-annexin-II antibodies have been found to prevent cytomegalovirus plaque formation [Additionally, several of the inserted mutations encode proteins that are thought to participate in trafficking of cargo in cells, and may participate in various stages of virus infection. These include mutations in genes encoding three annexins (Anxa1, Anxa2, Anxa3) and calcyclin (ch other -18 – andormation . Annexinormation ) [17,18,ormation .Jam)-1 [One of the clones has a disruption of a novel cell receptor, OL-16, which is a member of the immunoglobulin superfamily ,22. A prJam)-1 , whereasJam)-1 . OL-16 iJam)-1 . Forced Cstf 64 KaD subunit, which affects polyadenylation of mRNA, can be cross-linked to the mRNA of herpes simplex mRNA in infected HeLa cell extracts [Pts), a major regulator of interferon activity [Several of the candidate genes have products that interact with either reovirus or with other viruses (Supplementary Table see ). Cellulextracts . Other cextracts -30. For activity  associatactivity ,33.Ppm1a [Madh7 [Ube1c [Map3k7ip1 [Mapkbp1 and the proteosome [N-linked protein glycosylation, a process that may affect compartmentalization of proteins or ligand interactions.Many of the targeted gene products have roles involving the Golgi or endosomal compartments Figure , and addPpm1a , Madh7 [a [Madh7 -37, Ube17 [Ube1c ,39, and ap3k7ip1 -43 (Tablap3k7ip1 ). In addoteosome . We haveoteosome ), and thlacZ reporter as an immediate-early gene [Eif3s10, AnxaI, Mgat1, and Igf2r genes, were resistant to HSV-I infection  gene was readily apparent 4 h following infection, whereas lacZ expression was barely detected in Eif3s10 mutant cells 16h following infection. In all cases, levels of lacZ expression correlated with susceptibility to HSV-1 infection, suggesting that resistance involved early steps in the viral lifecycle.As several of the genes listed in the Supplementary Table see  have beerly gene . Data res Figure , c and eCtcf, a transcriptional repressor of IGF-II, was directly responsible for reovirus-resistance. In particular, cells containing the Ctcf mutation express elevated levels IGF-II, while parental RIE1 cells forced to express IGF-II acquired high levels of reovirus resistance. The mutation in Ctcf was chosen for further analysis because it was one of 3 mutations affecting IGF-II expression and/or signalling [Candidate genes required for lytic reovirus infections were identified by tagged sequence mutagenesis, a process that permits rapid identification of genes disrupted by gene entrapment. Since virus-resistant mutants may arise by a variety of mechanisms, additional experiments are needed to demonstrate that individual genes disrupted by gene entrapment actually contribute to the reovirus-resistant phenotype. Even so, several lines of evidence suggest that the genes collectively identified by tagged sequence mutagenesis include cellular activities that participate in the virus lifecycle. First, reovirus-resistant clones were selected at higher frequencies from entrapment libraries than from non-mutagenized cells, suggesting that reovirus-resistant phenotypes were induced by gene trap mutagenesis. Second, the genes associated with reovirus resistance differed from genes targeted in an unselected manner in mouse ES cells. Known mutational hot spots of the U3NeoSV1 were under-represented, and a number of mutations associated with virus resistance appeared to cluster within specific cellular processes and/or affected different components of multi-protein complexes that are likely to play roles in the virus lifecycle. These include IGF-II expression/signalling (3 genes), cytoskeletal/vesicular/trafficking (20 genes), signalling pathways (11 genes), and apoptosis 4 genes). Finally, we recently demonstrated that the disruption of  genes. FIgf2r. Alternatively, mutations induced by gene entrapment may confer reovirus resistance as a result of gene dosage effects (e.g. haploinsufficiency). For example, recent data suggests that the most common genetic disease in Caucasians, cystic fibrosis, involves mutations in the ABC-cassette transporter protein, CFTR, that confer resistance to infection by Salmonella typhi [Cryptosporidium parum [Pgy1 (Abcb1), encodes P glycoprotein  to disrupt all expressed genes, it seems unlikely that all the genes that are required for virus infection, and that can be targeted by tagged sequence mutagenesis, were identified in the present study.The circumstances that allow gene entrapment to disrupt the function of diploid genes illustrate that events secondary to provirus integration may be required for expression of some reovirus resistant phenotypes. Consequently, while the entrapment libraries were theoretically large enough  and Anxa2 genes was accompanied by a dramatic increase in immediate-early gene expression. This temporal enhancement of HSV-1 replication may reflect activities of calcyclin and annexin 2 proteins that suppress HSV-1 entry [Genetic alterations giving rise to reovirus resistant clones have variable effects on HSV-1 replication, with some reovirus resistant clones showing enhanced HSV1 replication. The reasons for this are unknown, although each of these two viruses enter cells by different mechanisms. The early steps in HSV replication require entry into cells, release of the capsid with migration to the nucleus for which virus and cellular proteins play roles ,54, wher-1 entry -57.Anxa1, clones with mutations in Eif3s10, Mgat1, and Igf2r also show decreases in transcription and translation of virus mRNA and cell death. Of these, mutations in the Igf2r are known to affect HSV replication [Eif3s10, Anxa1, and Mgat1 are novel. These data suggest that some of the candidate genes discovered in clones surviving reovirus infection may affect common cellular processes that are used by other viruses.In addition to the clone with a mutation in lication ,54,58; wStudies in a variety of systems indicate that resistance to infection may be found in nature or achieved in cultured cells. Results presented here support the hypothesis that pathways involved in reovirus infection can be identified through a functional genomics approach based upon insertional mutagenesis. Systematic selection of virus-resistant mutant cells in which the mutant gene can be easily identified may also identify targets for the development of anti-viral therapies. Drugs that disrupt cellular processes may circumvent the problem of virus resistance, generally observed with drugs against virus-encoded proteins. Moreover, since mutations associated with virus resistance are, by necessity, not lethal to the cell, drugs that target the same processes are not expected to have overtly toxic side effects. The fact that the resulting candidate genes may play roles in the replication of other viruses, suggests that different viruses may use similar host proteins for common steps required for virus entry, disassembly, transcription, translation, and reassembly. This view is supported by our studies with HSV-1 and by published reports implicating several of the genes disrupted in reovirus resistant cells (Supplementary Table see ) in the 3 mammalian genes, the definition of function will become the major task facing scientists interested in the relationship between host genes and viral disease over the next decade.The dramatic increase in the pace of the genome project has led to an explosion of information concerning the sequence of the genome of several species of animals and pathogenic organisms. However, most of the gene sequences have not been functionally ascribed with regard to host-parasite interactions. As there are approximately 30 to 50 × 10Candidate host genes that participate in lytic virus infections were identified utilizing insertional mutagenesis. Mutant cell clones were recovered that lost their capacity to support virus replication, but were able to proliferate. There was enrichment for genes that were involved in particular metabolic or signalling pathways, with many of the genes being selected more than once from independently derived libraries of RIE-1 cells. Several of the gene products are known to bind to each other. These genes or their products, which are identified by this process of selection, may provide targets for therapeutic intervention.Reovirus type 1, strain Lang, was initially obtained from Bernard N. Fields. Virus was passaged in L-cells and a third passaged stock was purified over a CsCl gradient as previously described and was used for these experiments . To deve4 gene entrapment events, were expanded until approximately 103 sibling cells represented each mutant clone. These cells were plated at a sub-confluent density and incubated in serum-free media for 3 days until they became quiescent, and infected with reovirus serotype 1, MOI of 35 plaque forming units (pfu) per cell. Eighteen hours following infection, the cells were detached with trypsin, and plated in DMEM medium containing 10% fetal bovine serum (FBS) (Hyclone Laboratories, Inc., Logan, Utah, USA). After 6 hrs, the medium was removed and cells were maintained in serum-free medium until only a few cells remained attached to the flask. On average, one to ten clones were recovered from a library consisting of 107 mutant cells, an enrichment for selected cells of six orders of magnitude. Cells that survived the selection were transferred to cell culture plates in media containing 10% FBS and cells were divided for extraction of DNA and cryopreservation.Following infection of RIE-1 cells with the U3neoSV1 vector, MOI of 0.1, mutagenized cells were selected for neomycin resistance in medium containing 1 mg/ml G418 sulfate  . Twenty lacZ, was determined by standard northern blot techniques and β-galactosidase assay, respectively.The transcription and translation of the HSV-1 immediate early gene reporter gene, Libraries of mutagenized cells were infected with reovirus serotype-1, strain Lang, to select for clones resistant to lytic infection. Selection of virus-resistant clones was performed in serum-free medium to suppress the emergence of persistently infected (PI) cells . This is-5 and the sequence was non-repetitive. In most cases, the matching gene was in the same transcriptional orientation as the provirus. Moreover, matches involving cDNA sequences were co-linear across exons present in the flanking genomic DNA and diverged at splice sites. As indicated, virtually all of the genes identified had matches to murine, rat, or human gene sequences with p < 10-10.Genomic DNA immediately adjoining the 5' end of the proviral insert in each of 130 cell lines was cloned by plasmid rescue . ApproxiELO, and JS conducted most of the laboratory work. HER provided the vectors and advice on their use. DHR discovered that persistently infected cells require serum to survive, allowing the selection of genetically resistant cell clones, and did the genetic analysis. Drs. Ruley and Rubin provided funding and supervision for the research, and prepared the manuscript. All authors have read and approved the final manuscript.Genes associated with resistance to lytic reovirus infection identified by tagged sequence mutagenesis A list of previously named genes disrupted by the insertional mutagen, U3neoSV1, and recovered in cell clones resistant to lytic infection is provided. The rat mRNA and vector insertion site accession number, rat chromosome location, human homologue chromosome location, link to the NCBI Entrez Gene and NCBI Nucleotide databases, and known virus interactions are listed.Click here for file


The complexity of parasitic infections requires novel approaches to vaccine design. The versatility of DNA vaccination provides new perspectives. This review discusses the use of prime-boost immunizations, genetic adjuvants, multivalent vaccines and codon optimization for optimal DNA vaccine design against parasites. DNA vaccination was introduced in 1990 by a study that demonstrated the induction of protein expression upon direct intramuscular injection of plasmid DNA in myocytes -4. DNA vSuccessful vaccines should be able to induce strong immune responses which are long-lasting and in most cases providing protection against different strains of the same pathogen. Progress has been made towards development of DNA vaccines against viral and bacterial pathogens showing protection and lasting immunity . ApplicaCurrent sub-unit vaccines predominantly induce strong antibody responses and weak cellular immunity. DNA vaccines in animal models can induce both strong humoral and cellular mediated responses, but although safe in humans, DNA vaccines do not produce the same magnitude of cellular immunity . In casePlasmodium yoelii DNA vaccine, mice were primed intramuscularly with DNA vaccine and granulocyte/macrophage colony stimulating factor (GM-CSF) plasmid and boosted with rVv encoding the same circumsporozoite protein (CSP) [P. falciparum [P. berghei challenge characterized by strong CD8+ T cell responses was observed in mice after intradermal adenovirus-prime-MVA-boost encoding CSP [P. falciparum pre-erythrocytic TRAP antigen were administered via gene-gun to healthy volunteers with no adverse effects [P. falciparum. Another group demonstrated that priming with DNA vaccine for P. falciparum CSP and boosting with a recombinant protein vaccine in adjuvant (RTS, S/AS02A) induced the production of significant antibody and T cell responses in healthy volunteers [P. falciparum TRAP antigen is safe, well tolerated and induces responses greater than those observed in naive volunteers [To further improve the efficacy of a in (CSP) . This colciparum . Completding CSP . These s effects . The pol effects . Furtherlunteers . Phase Ilunteers . BoostinAdjuvants are used to strengthen the immune response to a vaccine and have been critical in modern vaccine development. Genetic adjuvants are expression vectors encoding biologically active molecules such as cytokines, chemokines and co-stimulatory molecules. These adjuvants can be encoded on the same vector as the antigen or expressed on a separate vector and co-injected with the vaccine. This method provides adjuvant activity at the site of antigen production, with lasting effect from transfected cells. Cytokines are chosen as genetic adjuvants because they regulate cells involved in host defense and can be used to modulate immune responses. Co-delivery of cytokines in DNA vaccine formulation has been used extensively for a wide range of infectious and parasitic diseases  and either mouse GM-CSF or IL-4 to determine their adjuvant effect in mice. GM-CSF may work as adjuvant through its activating effect on dendritic cells and macrophages. Intramuscular vaccination with Sm-p80 alone provided 39% protection and this protection was significantly increased to 44% with GM-CSF co-administration and 42% with IL-4. The addition of GM-CSF led to an increase in total IgG and IgG1 while Th1 type IgG2a antibody titers remained high in protected animals [Siddiqui et al.  generate animals . GreaterAnother advantage of DNA vaccines is the possibility to integrate several antigens into the plasmid or to administer a mixture of plasmid vectors. The development of multivalent vaccines consisting of several antigens is a novel approach to create broad range protection against different parasite strains and parasite life cycle stages (see Table Leishmania major-activated C kinase (LACK), thio-specific antioxidant (TSA), and L. major stress-inducible protein (LmST11) was able to induce complete and long lasting protection after parasite challenge in mice compared to killed Leishmania parasites and rIL-12 [L. major cysteine proteinase genes (Cpa/Cpb) was protective and characterized by IFN-γ production by spleen cells, while separate injections were not protective [Vaccination studies against leishmaniasis in mice have identified various parasite antigens with varying degrees of protection as protein vaccines. When combined into multivalent DNA vaccines these antigens have the ability to confer complete or enhanced protection. In fact, a DNA vaccine including a mixture of plasmids encoding three antigens, d rIL-12 . This prd rIL-12 . It was otective . The cysotective  as the nin vitro and antigen specific responses in vaccinated animals. In our laboratory, we have developed a codon-optimized DNA vaccine encoding a portion of the Entamoeba histolytica Gal-lectin [E. histolytica genes are rich in A:T codons, whereas mammalian codons are more G:C rich. Protein expression of the E. histolytica Gal-lectin protein using the wild type sequence was difficult and stable clones were difficult to obtain in mammalian cells. Codon optimization was performed to ultimately increase protein expression in gerbils, a model for experimental amoebiasis; therefore gerbil codon usage was used to re-write the Gal-lectin Hg1l gene. Transfection of Cos-7 cells with the optimized vaccine construct produced a protein which was immunoreactive with a Gal-lectin specific monoclonal antibody (3F4), demonstrating successful expression of this amoebic protein. Upon vaccination with this codon optimized DNA plasmid, mice developed antigen specific antibodies of the Th1 isotype and Gal-lectin specific cellular immune responses.Interspecies differences in codon usage are a major obstacle in DNA vaccine development. This is due to the fact that DNA vaccines use host cells for transcription and translation of proteins. Every species has a codon bias for which most genes are encoded and this use of selected codons is related to gene expression efficiency. Closely related species use similar codons. However, in cases where there is a great difference in codon usage between the pathogen and mammals, codon optimization may be required. This strategy involves the modification of codon usage for the genes encoded in a DNA vaccine to a suitable codon bias for increased expression in mammals. This method has proved effective in many systems ,49,50, il-lectin . E. histet al. [P. falciparum DNA vaccines when administered as a mixture of plasmids compared to single plasmid injections. Another study, however, demonstrated that a mixture of three plasmids encoding P. falciparum blood-stage antigens had no reduction in immunogenecity when co-injected [In this review, strategies for increased DNA vaccine efficacy against parasitic diseases to date, i.e. prime-boost immunizations, genetic adjuvants, multivalent vaccines and codon optimization, have been discussed. DNA vaccine technology provides the versatility required to separate protective components of immunity from counter-protective responses. As seen with genetic adjuvants, DNA vaccines can focus on the protective cytokines involved and include antigens that stimulate the production of specific cytokines. This allows designing vaccination strategies that are tailored to a particular infection or even a specific stage of infection. Parasitic diseases are complex, involving changes in immunological responses during the course of infection and changes in immunity to stage specific antigens. The advent of optimization strategies with DNA vaccines presents researchers with the tools to design effective vaccines with specific purposes. It is possible to enhance DNA vaccine efficacy, thus increasing immune responses and protection, through the use of these methodologies. However, it is important to note that these strategies need to be adjusted to the parasite system in order to provide the greatest benefit upon vaccination. For example, Sedegah et al.  reportedinjected . Therefoinjected ,24,32,54® XR1 is a needle-free powder injection system that delivers fine gold particles coated with the vaccine vectors directly into epidermal cells, specifically dendritic cells. This vaccination method is advantageous since it eliminates the cold chain requirement and reduces the chances of needle-borne contamination. Moreover, the gene-gun method is safe and seems as immunogenic as intramuscular injections in these trials [The method of vaccine delivery is an important variable in vaccination design. DNA vaccination has been successful through a variety of injection routes, including intradermal, intramuscular, and intranasal. Although intramuscular injections are most common and give consistent responses, alternative routes of delivery may be desired depending on the disease model. Mucosal DNA vaccine immunizations, against intestinal parasites for example, are effective to generate mucosal immune responses at the site of infection. For leishmaniasis, where the disease manifests itself as cutaneous lesions, an intradermal injection targeting Langerhans' cells may be optimal . The gene trials .Leishmania parasites in mice have improved cellular immune responses when plasmids expressing IL-12 and IL-18 are co-injected [P. cynomolgi antigens (CSP/TRAP) resulted in lower peak parasitemia and higher antibody and cellular responses than controls [The greatest challenge in designing DNA vaccines against parasites is making the vaccine suitable for humans while providing strong, long lasting immune responses. Many studies in laboratory animals are successful but the results cannot be replicated in humans. The prime-boost strategy has shown the most success as a delivery technique in larger animals or humans. Field trials with prime-boost malaria vaccines are ongoing and will provide experts with insight with regards to the safety and the immune responses required for protection in humans. Meanwhile, other groups are reporting improved responses in mice or larger mammals with other vaccines, suggesting that this vaccination strategy may be applicable to many other parasitic diseases ,29. A vainjected . A DNA pcontrols . Taken tThe author(s) declare that they have no competing interests.CI and KC produced the manuscript together. All authors read and approved the final manuscript.


Hematopoiesis is a complex developmental process controlled by a large number of factors that regulate stem cell renewal, lineage commitment and differentiation. Secreted proteins, including the hematopoietic growth factors, play critical roles in these processes and have important biological and clinical significance. We have employed representational difference analysis to identify genes that are differentially expressed during experimentally induced myeloid differentiation in the murine EML hematopoietic stem cell line.Ciona intestinalis and the invertebrate worm Caenorhabditis elegans and two genes in two insect species, Drosophila melanogaster and Anopheles gambiae. Six FAM20 family members were identified in the genome of the pufferfish, Fugu rubripes and five members in the zebrafish, Danio rerio. The mouse Fam20a protein was ectopically expressed in a mammalian cell line and found to be a bona fide secreted protein and efficient secretion was dependent on the integrity of the signal sequence. Expression analysis revealed that the Fam20a gene was indeed differentially expressed during hematopoietic differentiation and that the other two family members (Fam20b and Fam20c) were also expressed during hematcpoiesis but that their mRNA levels did not vary significantly. Likewise FAM20A was expressed in more limited set of human tissues than the other two family members.One identified clone encoded a previously unidentified protein of 541 amino acids that contains an amino terminal signal sequence but no other characterized domains. This protein is a member of family of related proteins that has been named family with sequence similarity 20 (FAM20) with three members (FAM20A, FAM20B and FAM20C) in mammals. Evolutionary comparisons revealed the existence of a single FAM20 gene in the simple vertebrate The FAM20 family represents a new family of secreted proteins with potential functions in regulating differentiation and function of hematopoietic and other tissues. The Fam20a mRNA was only expressed during early stages of hematopoietic development and may play a role in lineage commitment or proliferation. The expansion in gene number in different species suggests that the family has evolved as a result of several gene duplication events that have occurred in both vertebrates and invertebrates. Hematopoietic differentiation is a complex process whereby multiple functionally and morphologically distinct cell types arise from a population of pluripotent hematopoietic stem cells (PHSCs) . The accWe have used a pair of factor-dependent murine cell lines to identify novel genes expressed within distinct hematopoietic lineages as an approach to the identification of novel candidate genes for development of diagnostic and therapeutic approaches to leukemia. The EML and MPRO cell lines were both established by infecting murine bone marrow cells with a retrovirus expressing a dominant negative retinoic acid receptor α (RARα) protein ,5. The iWe have employed the representation difference analysis technique  to identTotal RNA was prepared from EML cells grown in the presence of SCF alone (0 hour) or in medium supplemented with IL-3 and atRA for 72 hours. The RNA was converted to cDNA and subjected to three rounds of RDA as previously described . Six difThe initial cDNA isolated by RDA was a 273 bp fragment that appeared to contain the coding sequence of the C-terminus of a protein that was not present at that time in public databases. To identify the full open reading frame of this cDNA, we first performed rapid amplification of cDNA ends (RACE) in both the 5' and 3' directions. Extension in the 3' direction revealed the presence of a consensus polyadenylation signal located 154 nucleotides downstream of the putative translation stop codon. Extension in the 5' direction yielded an additional 443 bp of sequence containing a contiguous ORF. Comparison of this extended sequence to public databases identified a cDNA (NM_017565) that was identical to clone 1623 in the region of overlap. This cDNA was isolated from a mouse mammary tumor but no functional analysis had been performed. We designed PCR primers based on the published sequence and confirmed that the cDNA isolated from EML cells was identical to the published sequence. The full length ORF was 1623 bp in length and encoded a protein of 541 amino acids Figure . The pronamed four-jointed [The full length cDNA and the encoded protein were compared to sequences in public databases. We reported previously a weak similarity to a protein named Fjx1, which is the mouse orthologue of a Drosophila protein -jointed . HoweverCiona intestinalis is a model for a basal chordate organism and has emerged as a powerful model for evolutionary and developmental studies [C. intestinalis and thus the identification of an orthologue in this organism can provide useful information for evaluating the evolutionary origin of the members of a gene family. Consistent with this concept, we identified a single cDNA and the corresponding genomic locus in C. intestinalis that displayed significant sequence similarity to the mammalian FAM20 genes and proteins (Table Drosophila melanogaster and Anopheles gambiae with one family member in Caenorhabditis elegans (Table Fugu rubripes) and zebrafish (Danio rerio) revealed the presence of six and five family representatives, respectively  was compared and revealed obvious inter and intra orthologue similarities figure . Each FAC. intestinalis clustered with FAM20B proteins, suggesting that this represents the ancestral branch of the FAM20 family. A single protein from Fugu and zebrafish clustered with the FAM20A and FAM20B family members while two Fugu and two zebrafish proteins clustered with FAM20C members. However, two Fugu proteins and one zebrafish protein clustered on a separate branch between FAM20A and FAM20B. In order to determine the subfamily to which these proteins belonged, we made use of the high degree of conservation of exon size and number noted in the mammalian genes  and either retained there or processed and transported into the Golgi apparatus and secreted from the cell. Many proteins are glycosylated during their transit through the ER and Golgi apparatus and the mouse Fam20a protein contains four potential sites for N-glycosylation (indicated in red type in figure 14–Leu15 to Asp-Glu) were predicted to disrupt the signal sequence without grossly altering the protein structure. Again the mutant protein displayed nuclear staining and was absent from the ER . Therefore, we examined the expression patterns of the three genes in a panel of cDNAs derived from various human tissues. FAM20A displayed the most restricted expression pattern with high levels in lung and liver and intermediate levels in thymus and ovary Figure . Low levSeveral classes of secreted proteins, including the colony stimulating factors or hematopoietins, are important regulators of hematopoietic differentiation and function . These mAt the present time, the specific function(s) of the FAM20 proteins is unknown. Analysis of the sequences of the proteins from various species failed to reveal obvious similarities to known functional domains except for an N-terminal signal sequence. Expression studies clearly demonstrated that the mouse Fam20a protein is a secreted protein and that disruption of the signal sequence prevented the detection of the glycosylated form of the protein in cell media. Surprisingly, disruption of the signal sequence resulted in redistribution of intracellular Fam20a from a cytoplasmic compartment that is likely to be the ER to the nucleus. It is unclear whether this redistribution is functionally significant and is presumably due to the presence of a cryptic nuclear localization signal (NLS) in the protein. NLSs are generally comprised of stretches of basic residues  and seveOverall, the FAM20 proteins vary in length between 400–670 amino acids with the FAM20B family members being the shortest and the FAM20C members generally being the longest. The variation in length is due to differences in the length of the less conserved N-terminal region. The FAM20 proteins are further characterized by a highly conserved C-terminal region of approximately 350 amino acids that we named the conserved C-terminal domain (CCD). We noted the presence of three regions that are more highly conserved amongst all family members, with the most invariant extended sequence being the DRHHYE heptapeptide within conserved region 2. The function of the peptide is as yet unknown but three possible functions can be proposed. First, the histidines within this sequence could be involved in the coordination of metal ions that may be required for FAM20 protein function. Second, FAM20 proteins may be enzymes and this sequence may be a component of the active site of the enzyme. Interestingly, a weak match was detected between this region of certain FAM20 family members and a conserved domain within the phosphatidylinositol 3- and 4-kinases ; howeverD. melanogaster and A. gambiae), a simple chordate , three mammals (H. sapiens, M. musculus and R. norvegicus) and two fish (F. rubripes and D. rerio). In each case, the identification of these genes as bona fide transcription units is supported either by direct experimental evidence for the human and mouse genes, or by the existence of multiple EST sequences in public databases. A single FAM20 gene was identified in C. intestinalis, which is considered to be a representative of a basal chordate related to the common ancestor of humans and other higher chordates [C. intestinalis genome encodes approximately 16,000 genes and generally contains single representatives of superfamilies in higher vertebrates, thereby permitting the elucidation of likely evolutionary origins of genes within these families [Tremendous progress has been made over the past decade in the sequencing of genomes from species at different positions on the evolutionary tree -25. Thishordates . The C. families . The C. families . This pafamilies . In the A further round of gene duplication has been proposed to have occurred in fish  and the Expression analysis of the three FAM20 family members in mammalian tissues and hematopoietic cells showed that FAM20A is expressed in a much more restricted pattern that the other two members. Importantly, Fam20a was also the only member to display obvious differential expression in hematopoietic cells undergoing myeloid differentiation. Fam20a mRNA levels were highest during intermediate stages of differentiation of EML cells, at a time when many cells are becoming committed to the myeloid lineages and undergoing extensive proliferation ,8,29. EM-5 M, Sigma) for 72 hours. Terminal granulocytic differentiation was induced by removing IL-3 and SCF and adding 10% BHK-HM5 CM as a source of granulocyte/macrophage-colony stimulating factor (GM-CSF). MPRO cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan, UT) and 10% BHK-HM5 CM. Terminal differentiation was induced by adding 1 – 10-5 M atRA to the culture medium. For collection of RNA samples at different timepoints, 10 cm dishes were seeded with 150,000 cells and cultured under identical conditions. Cells were harvested from individual wells at 24 hour intervals for RNA preparation.EML and MPRO cells were cultured as described previously ,30. Brie+ RNA prepared from EML cells at zero hours (stem cell stage) and 72 hours (promyelocyte stage) was converted to double stranded cDNA and three rounds of RDA was performed as described previously [Total RNA was prepared from isolated cells using a modified guanidium isothiocyanite/phenol extraction procedure as described previously . Poly A+eviously . cDNA cleviously ,8 and cl4 per well in six well plates and transfected using the Effectene Transfection Reagent  under conditions recommended by the manufacturer.Cos-1 cells were maintained in Dulbecco's modified Eagle's medium  containing 10% FBS and Penicillin/Streptomycin (BioWhittaker) under standard cell culture conditions. For transfections, cells were plated at a density of 2.5 × 10Analysis of the sequence of the original 1623 cDNA isolated from the RDA experiments revealed that it contained the coding sequence from the C-terminus of an uncharacterized protein. 5' and 3' rapid amplification of cDNA ends (RACE) was performed using Marathon-Ready cDNA from mouse spleen using the Advantage cDNA PCR kit (both from Clontech) as described previously . The RAC. Genome specific searches were performed either through NCBI or Ensembl  or through genome specific websites . Searches were performed either as nucleotide-nucleotide (blastn) searches or as protein-translated nucleotide (tblastn) searches. In general, these searches were sufficient to identify gene regions encoding the most highly conserved regions of the proteins, particularly in the fish and Ciona genomes. To assemble complete genes, genomic regions containing the identified regions of similarity were downloaded into VectorNTI and searches were performed for individual exons, initially derived from mammalian genes but subsequently, from potential orthologues in fish or lower vertebrates, depending on the sequence being examined. Putative exons were examined for the presence of consensus splice donor and acceptor sites and complete sets of exons were assembled into cDNAs for translation and further comparisons. These results were compared against genes assembled by two gene prediction programs, FGENESH:  and GENSCAN: , however, these programs often made incorrect assignments that required manual assessment of the results. The protein sequences derived from each of the examined species were primarily used in the comparisons to define subfamily assignments and alignments were performed in the AlignX program in VectorNTI using the blosum62 scoring matrix. Protein domain searches were performed using the Simple Modular Architecture Research Tool (SMART)  and/or Profilescan . Signal sequence searches were performed using the SignalP analysis program [All basic sequence manipulations were performed using the VectorNTI suite of sequence analysis programs (Informax/Invitrogen Corp., Carlsbad, CA). Identification of FAM20 members from different species was initially achieved using standard BLAST searches at NCBI  program . SequencXhoI and HindIII restriction sites were engineered into the PCR primers . Similar strategy was used to clone Fam20a and Fam20aΔ23 in the pcDNA3.1 vector . The Fam20a(SSmut) was generated using a PCR-based site-directed mutagenesis kit (Quickchange, Stratagene). All of the sequences were verified by sequencing using an ABI automated sequencer.The Fam20a and Fam20aΔ23 were constructed in the pEGFP-N1 vector by PCR cloning. Whole cell extracts were prepared from transfected COS-1 cells by lysing directly in 2X Laemmli Sample Buffer as described previously . Purifie2PO4-H2O (pH 8.0), 150 mM NaCl, 10 mM Imidazole). His-tagged proteins were purified using Ni-NTA Magnetic Agarose Beads  according to manufacturer's recommendations. Following the purification step they were either used directly in western blot analysis or for deglycosylation experiments. Purified proteins were subjected to enzymatic deglycosylation using the GlycoPro deglycosylation kit (ProZyme, San Leandro, CA) following the manufacturer's protocol.COS-1 cells were transfected as described above and media were collected after 48 hours and mixed with freshly made 10X binding buffer (500 mM NaH2HPO4 (pH 7.3)) containing 1% Triton X-100 for 10 minutes to permeabilize the cell membranes. They were then transferred to PBST (PBS containing 0.1% Tween-20) and incubated for 30 minutes. Mouse monoclonal anti-Myc antiserum (Santa Cruz) was used at 1:2,000 dilution in PBST containing 1% bovine serum albumin (1 hour at room temperature). Coverslips were then washed with PBST three times and incubated with the secondary antibody (Alexa Fluor 488 [Molecular Probes, Eugene, OR]) for 1 hour. Coverslips were washed three times in PBST and once in water for 15 minutes each, and were mounted on glass slides. The cells were observed by epifluorescence microscopy using an Axiovert 135 TV microscope (Zeiss, Gottingen, Germany). Images were captured with a Kodak DC290 zoom camera and analyzed with MetaMorph 6.0 .COS-1 cells were plated on glass coverslips and transfected with the indicated constructs the following day. After 48 hours of incubation, the cells were fixed in 1% formaldehyde for 30 minutes at room temperature. Coverslips were incubated in PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na- Reverse Transcriptase (Invitrogen, Carlsbad, CA), 0.125 mM dNTPs, 10 mM DTT, and 1 μM oligo (dT)15 in a total volume of 50 μl for 1 hour at 37°C. 2 ul of the RT reaction was then mixed with 1 ul of 10X PCR Buffer (500 mM Tris-HCl (pH8.3), 2.5 mg/ml crystalline BSA and MgCl2 at 10, 20 or 30 mM , 0.2 mM each dNTP, 0.05 μM of each primer and 1.25 Units Taq DNA polymerase (Fisher Scientific, Pittsburgh, PA) in a total volume of 10 μl. Reactions were loaded into a capillary tube and PCR cycles were carried out using the Rapidcycler Thermal cycler (Idaho Technology). Annealing temperatures and Mg2+ concentrations were initially optimized for each primer. A commercial set of tissue cDNAs, (Human Multiple Tissue cDNA (MTC) Panels, Clontech, Palo Alto, CA) was used for the RT-PCR analysis of human FAM20 family members.Total RNA was purified as described above from EML and MPRO cells at 24 hour timepoints during the differentiation process of each cell line The primers were designed using the Vector NTI sequence analysis suite of programs (InforMax, Frederick, MD) and were follows: MmFam20a forward 5'-catagaggcccacggcgagcg-3' and reverse 5'-atggaatggggcaacag gggc-3'; Mmfam20b forward 5'-tggacaggattctgggtttc-3' and reverse 5'-ccagggatgtcgatgtttct-3'; MmFam20c forward 5'-agcagacgagagagcaggag-3' and reverse 5'-cggatctccttggtcatgtt-3'; HsFAM20a forward 5'-ctggcaggaaaagagtg-3' and 5'-cccgaacttggtgaacatct-3', HsFAM20b forward 5'-ccctgaagagacaccagaagagc-3' and reverse 5'-gaaacccagaatcctgtcca-3', HsFAM20c forward 5'-ggctcacgttccacattggt-3' and reverse 5'-aaagtcagggggtgtctcct-3'.; mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward 5'-aatggtgaaggtcggtg tgaac-3' and reverse 5'-gaagatggtgatgggcttcc-3'; human (GAPDH) forward 5'-tgaaggtcggagtcaacggatttggt-3' and reverse 5'-catgtgggccatgaggtccaccac-3'. GAPDH was used as control for cDNA integrity. Single stranded cDNA was reverse transcribed from 2 μg of total RNA using 400 Units of Superscript RNase HFAM20: family with sequence similarity 20; EML: Erythroid, myeloid, lymphoid cell line; MPRO: mouse promyelocyte cell line; GM-CSF: granulocyte/macrophage-colony stimulating factor; PHSC: pluripotent hematopoietic stem cell; HGF: hematopoietic growth factor; RAR: retinoic acid receptor; atRA: all trans retinoic acid; SCF: stem cell factor; IL-3: interleukin-3; RDA: representational difference analysis; RACE: rapid amplification of cDNA ends; CCD: conserved C-terminal domain; GFP: green fluorescent protein; CM: conditioned medium.DN isolated full length Fam20a cDNA, performed transfection experiments and drafted the manuscript. HY performed RT-PCR assays. IN assisted in construction of expression vectors. SS performed genomics analyses. EC and EGB provided reagents and assistance in performing RT-PCR assays. YD performed the original representational difference analysis and SCW directed the project and performed genomics analyses. SCW also wrote the final draft of the manuscript and all authors read and approved this version.