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In Italy, the inclusion of measles vaccine in children immunization schedule and the promotion of national mass vaccination campaigns increased measles vaccination coverage. However, measles outbreaks continue to occur increasingly involving adolescents and adults. The aim of this study was to evaluate the prevalence to measles antibody in a sample of Italian population between 1993 and 2018. Human serum samples from subjects aged 3–40 years were collected between 1993 and 2018 and tested for measles IgG antibodies by commercial ELISA. During the study period, the 3–10-year-old age group showed the most important change, with a significant increase in 2003–2007 in both seroprevalence and IgG levels, followed by a slow decrease. The 11–18-year-old age group showed relatively stable seroprevalence rates and IgG levels over the years. The 19–30-year-old group showed stable seroprevalence rates, albeit with a decrease in IgG levels. After a significant increase in 1999–2002, the 31–40-year-old age group had high seroprevalence rates and IgG levels. Despite efforts at national level for implementing measles vaccination, a large proportion of the population is still susceptible to measles. Even if vaccination coverage increases enough to achieve the level of immunization required for herd immunity in new birth cohorts, outbreaks will continue to occur if there are immunity gaps in older age groups. Establishing policies for measles vaccination targeting adult population is needed to close immunity gaps and reach the elimination goal. [ABSTRACT FROM AUTHOR]

Background: The immunological response to influenza vaccine and/or natural infection is evaluated by serological techniques, the most common being hemagglutination inhibition (HI), single radial hemolysis (SRH), and virus neutralization assays, which is commonly used in a micro‐neutralization (MN) format. ELISA is not officially required; however, this assay is able to measure different class‐specific antibodies. The four assays identify different sets or subsets of antibodies. Objectives: The aim of this study was to establish the correlation among four serological assays using four seasonal influenza strains. Methods: The HI, SRH, MN assays, and ELISA were performed on four seasonal influenza strains. Results: A strong positive correlation was found between HI and MN and between SRH and MN assays for influenza A strains. The B strains also showed good correlations among the three assays. A positive correlation was also found between ELISA and the "classical" assays for all strains. Concerning the correlates of protection, as defined by HI ≥ 40 and SRH ≥ 25 mm2, good agreement was observed for the influenza A strains. By contrast, the agreement for the B strains was very low. Conclusions: There is a positive strong correlation among the four serological assays for both A and B strains, especially for the HI and MN assays. There is good agreement on correlates of protection between HI and SRH assays for the A strains, but very low agreement for the B strains, suggesting higher sensitivity of SRH than HI assay in detecting antibodies against the influenza B viruses. [ABSTRACT FROM AUTHOR]

Plasmodium falciparum: apical membrane antigen 1 (AMA1) is a candidate malaria vaccine antigen expressed on merozoites and sporozoites. The polymorphic nature of AMA1 may compromise vaccine induced protection. The humoral response induced by two dosages (10 and 50 mg) of a single allele AMA1 antigen (FVO) formulated with Alhydrogel, Montanide ISA 720 or AS02 was investigated in 47 malaria-naïve adult volunteers. Volunteers were vaccinated 3 times at 4 weekly intervals and serum samples obtained four weeks after the third immunization were analysed for (i) Antibody responses to various allelic variants, (ii) Domain specificity, (iii) Avidity, (iv) IgG subclass levels, by ELISA and (v) functionality of antibody responses by Growth Inhibition Assay (GIA). About half of the antibodies induced by vaccination cross reacted with heterologous AMA1 alleles. The choice of adjuvant determined the magnitude of the antibody response, but had only a marginal influence on specificity, avidity, domain recognition or subclass responses. The highest antibody responses were observed for AMA1 formulated with AS02. The Growth Inhibition Assay activity of the antibodies was proportional to the amount of antigen specific IgG and the functional capacity of the antibodies was similar for heterologous AMA1-expressing laboratory strains. [ABSTRACT FROM AUTHOR]

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) is a leading blood stage vaccine candidate. Plasmodium knowlesi AMA1 (PkAMA1) was produced and purified using similar methodology as for clinical grade PfAMA1 yielding a pure, conformational intact protein. Combined with the adjuvant CoVaccine HTTM, PkAMA1 was found to be highly immunogenic in rabbits and the efficacy of the PkAMA1 was subsequently tested in a rhesus macaque blood-stage challenge model. Six rhesus monkeys were vaccinated with PkAMA1 and a control group of 6 were vaccinated with PfAMA1. A total of 50 μg AMA1 was administered intramuscularly three times at 4 week intervals. One of six rhesus monkeys vaccinated with PkAMA1 was able to control parasitaemia, upon blood stage challenge with P. knowlesi H-strain. Four out of the remaining five showed a delay in parasite onset that correlated with functional antibody titres. In the PfAMA1 vaccinated control group, five out of six animals had to be treated with antimalarials 8 days after challenge; one animal did not become patent during the challenge period. Following a rest period, animals were boosted and challenged again. Four of the six rhesus monkeys vaccinated with PkAMA1 were able to control the parasitaemia, one had a delayed onset of parasitaemia and one animal was not protected, while all control animals required treatment. To confirm that the control of parasitaemia was AMA1-related, animals were allowed to recover, boosted and re-challenged with P. knowlesi Nuri strain. All control animals had to be treated with antimalarials by day 8, while five out of six PkAMA1 vaccinated animals were able to control parasitaemia. This study shows that: i) Yeast-expressed PkAMA1 can protect against blood stage challenge; ii) Functional antibody levels as measured by GIA correlated inversely with the day of onset and iii) GIA IC50 values correlated with estimated in vivo growth rates. [ABSTRACT FROM AUTHOR]

Background: A DNA prime, poxvirus (COPAK) boost vaccination regime with four antigens, i.e. a combination of two Plasmodium knowlesi sporozoite (csp/ssp2) and two blood stage (ama1/msp142) genes, leads to self-limited parasitaemia in 60% of rhesus monkeys and survival from an otherwise lethal infection with P. knowlesi. In the present study, the role of the blood stage antigens in protection was studied in depth, focusing on antibody formation against the blood stage antigens and the functionality thereof. Methods: Rhesus macaques were immunized with the four-component vaccine and subsequently challenged i.v. with 100 P. knowlesi sporozoites. During immunization and challenge, antibody titres against the two blood stage antigens were determined, as well as the in vitro growth inhibition capacity of those antibodies. Antigen reversal experiments were performed to determine the relative contribution of antibodies against each of the two blood stage antigens to the inhibition. Results: After vaccination, PkAMA1 and PkMSP119 antibody titres in vaccinated animals were low, which was reflected in low levels of inhibition by these antibodies as determined by in vitro inhibition assays. Interestingly, after sporozoite challenge antibody titres against blood stage antigens were boosted over 30-fold in both protected and not protected animals. The in vitro inhibition levels increased to high levels (median inhibitions of 59% and 56% at 6 mg/mL total IgG, respectively). As growth inhibition levels were not significantly different between protected and not protected animals, the ability to control infection appeared cannot be explained by GIA levels. Judged by in vitro antigen reversal growth inhibition assays, over 85% of the inhibitory activity of these antibodies was directed against PkAMA1. Conclusions: This is the first report that demonstrates that a DNA prime/poxvirus boost vaccination regimen induces low levels of malaria parasite growth inhibitory antibodies, which are boosted to high levels upon challenge. No association could, however, be established between the levels of inhibitory capacity in vitro and protection, either after vaccination or after challenge. [ABSTRACT FROM AUTHOR]

Abstract: Plasmodium falciparum apical membrane antigen 1 (PfAMA1) is a leading asexual blood stage vaccine candidate for malaria. In preparation for clinical trials, PfAMA1 ectodomain (amino acid 25–545, FVO strain) was produced in Pichia pastoris by 35L scale fed batch fermentation under current Good Manufacturing Practice (cGMP). Fermentation was followed by a three-step chromatographic purification procedure resulting in a yield of 5.8g of purified protein. As judged by size exclusion chromatography, the cGMP-product comprised >95% PfAMA1 monomer, the remainder being predominantly PfAMA1 dimer. In SDS-PAGE two main bands of 68 and 70kDa and some minor bands were evident. Under reducing conditions a site of limited proteolytic cleavage within a disulphide bonded region became evident; less than 15% of the protein had this internal cleavage. By mass-spectrometric analysis, all bands analyzed in overloaded SDS-PAGE gels comprised PfAMA1 derived products. The protein was quantitatively bound by immobilized 4G2, a monoclonal antibody reactive with a reduction sensitive conformational determinant. The lyophilized product was stable for over 1 year. Immunopotency did not diminish, and storage did not lead to alterations in the behaviour of the protein upon formulation with adjuvants selected for Phase I clinical evaluation. These formulations also showed no pharmacotoxicity in rabbits. The final product conformed to preset criteria and was judged suitable for use in human clinical trials. [Copyright &y& Elsevier]

The major attractions of vaccines based on viral carriers (vectors) include their immunogenicity without adjuvant and the relative simplicity of their associated production processes in comparison with recombinant protein-based approaches. Two influenza virosomal vaccines – for influenza and hepatitis A – are registered for human use, and the virosome platform is being evaluated as the carrier for a Plasmodium falciparum vaccine that targets both the exo-erythrocytic and erythrocytic stages. Although safe and immunogenic, the first such virosome-based malaria vaccine showed no protection in a Phase IIa clinical trial. Nevertheless, the established safety profile of virosomes and their flexibility with regard to antigen delivery – allowing for antibody induction via the conjugation of peptides and T-cell induction via encapsulation – indicate that they warrant further exploration. [Copyright &y& Elsevier]

Apical membrane antigen 1 (AMA1) is a micronemal protein of apicomplexan parasites that appears to be essential during the invasion of host cells. Immune responses to Plasmodium AMA1 can have profound parasite-inhibitory effects, both as measured in vitro and in animal challenge models, suggesting AMA1 as a potential vaccine component. However, AMA1 is polymorphic, probably as a result of immune selection operating on an important target of naturally occurring immunity. The current understanding of AMA1 will be presented, particularly in relation to the vaccine potential of AMA1 and the approaches being taken towards clinical development. [Copyright &y& Elsevier]

Objectives: To investigate whether a low peripheral blood lymphocyte count is associated with increased mortality risk in older persons and to determine whether this association could be ascribed to ill health. Design: A cohort study with a total follow-up period of 1,602 person years. Setting: Leiden, the Netherlands. Participants: Four hundred thirty-six community-dwelling residents aged 85 and older. Measurements: Health status and leukocyte total and differential counts were assessed at baseline. Lymphocyte subsets were measured with a fluorescence-activated cell sorter. Age- and sex-adjusted mortality risks were estimated using Cox proportional hazard regression analysis. Results: There was no association between lymphocyte count and mortality in persons with ill health (mortality risk lowest vs highest quartile=1.16; 95% confidence interval (CI)=0.85–1.58, P =.35), but mortality was dependent on lymphocyte count if disease was excluded (mortality risk lowest vs highest quartile=2.14; 95% CI=1.08–4.23, P =.03). A similar increase in mortality risk was found when the cluster designation (CD)4[sup +] , CD8[sup +] , and CD16[sup +] lymphocyte subsets were analyzed. Within individuals, low values of the lymphocyte subsets were related and there was no compensatory increase in CD16[sup +] lymphocyte counts. A low lymphocyte count was not associated with specific causes of death. Conclusion: A low lymphocyte count was associated with an increased mortality risk in older persons without apparent disease. This association was not only found for the total lymphocyte count but also for the CD4[sup +] , CD8[sup +] , and CD16[sup +] lymphocyte subset counts. [ABSTRACT FROM AUTHOR]

Abstract: Attempts have been made at finding ways of cleaning used protein G agarose to revive their efficiency and make them reusable for purifying immunoglobulin G (IgG) in affinity column chromatography. A successful cleaning procedure that involved the use of 4-mol/L urea with 0.1-mol/L sodium hydroxide has been previously evaluated although it had some deleterious effect on the column binding capacity and compromised the resin efficiency. Efforts to develop base-tolerant affinity columns by using genetically engineered ligands such as the recombinant protein A from GE Healthcare have achieved some progress, with the column efficiency getting compromised to a lesser extent. However, genetically engineered ligands are even more expensive and may not be readily affordable in modest laboratory settings, especially if large-scale purchases are needed in routine use. We report here a novel and simple cleaning method involving the use of polyethylene glycol (PEG8000) that renews matrix-binding capacity comparable to a new resin while retaining high selectivity for IgG. [Copyright &y& Elsevier]

SARS-CoV-2 pandemic is causing high morbidity and mortality burden worldwide with unprecedented strain on health care systems. To investigate the time course of the antibody response in relation to the outcome we performed a study in hospitalized COVID-19 patients. As comparison we also investigated the time course of the antibody response in SARS-CoV-2 asymptomatic subjects. Study results show that patients produce a strong antibody response to SARS-CoV-2 with high correlation between different viral antigens (spike protein and nucleoprotein) and among antibody classes (IgA, IgG, and IgM and neutralizing antibodies). The antibody peak is reached by 3 weeks from hospital admission followed by a sharp decrease. No difference was observed in any parameter of the antibody classes, including neutralizing antibodies, between subjects who recovered or with fatal outcome. Only few asymptomatic subjects developed antibodies at detectable levels. [ABSTRACT FROM AUTHOR]

Plasmodium vivax malaria is increasingly recognized as a major global health problem and the socio-economic impact of P.vivax- induced burden is huge. Vaccine development against P. vivax malaria has been hampered by the lack of an in vitro culture system and poor access to P. vivax sporozoites. The recent generation of Plasmodium falciparum parasites that express a functional P. vivax AMA1 molecule has provided a platform for in vitro evaluation of PvAMA1 as a potential blood stage vaccine. Three so-called PvAMA1 Diversity Covering (DiCo) proteins were designed to assess their potential to induce a functional and broad humoral immune response to the polymorphic PvAMA1 molecule. Rabbits were immunized with the mixture of three, Pichia-produced, PvAMA1 DiCo proteins, as well as with 2 naturally occurring PvAMA1 alleles. For these three groups, the experimental adjuvant raffinose fatty acid sulfate ester (RFASE) was used, while in a fourth group the purified main mono-esterified constituent (RSL10) of this adjuvant was used. Animals immunized with the mixture of the three PvAMA1 DiCo proteins in RFASE showed high anti-PvAMA1 antibody titers against three naturally occurring PvAMA1variants while also high growth-inhibitory capacity was observed against P. falciparum parasites expressing PvAMA1. This supports further clinical development of the PvAMA1 DiCo mixture as a potential malaria vaccine. However, as the single allele PvAMA1 SalI-group showed similar characteristics in antibody titer and inhibition levels as the PvAMA1 DiCo mixture-group, this raises the question whether a mixture is really necessary to overcome the polymorphism in the vaccine candidate. RFASE induced strong humoral responses, as did the animals immunized with the purified component, RSL10. This suggests that RSL10 is the active ingredient. However, one of the RSL10-immunized animal showed a delayed response, necessitating further research into the clinical development of RSL10. [ABSTRACT FROM AUTHOR]

Simple Summary: In the present study, hematological and serum biochemical reference intervals were established for common marmosets sedated with alphaxalone through all life stages, including pregnancy and contraception. Clinically relevant changes were established for multiple parameters when comparing data based on age, sex, housing condition, contraceptive use, and pregnancy. The availability of updated reference intervals, calculated using a large sample size and using the appropriate statistical methods, is an essential part of providing optimum care and support to marmosets used in biomedical research. Marmosets are routinely used in biomedical research, therefore there is an increasing need for updated reference intervals calculated using a large sample size, correct statistics, and considering different variables. Hematological and biochemical values from 472 healthy common marmosets sedated with alphaxalone were collected over a ten-year period (2013–2023). The variables assumed to have influenced the blood-based parameters were compared, i.e., sex, age, housing condition, pregnancy, and contraceptive use. Reference intervals were calculated based on observed percentiles without parametric assumptions, and with parametric assumptions following Box–Cox transformation. Juvenile marmosets showed increased ALP, phosphate, WBC, lymphocyte count, and basophil count and decreased levels of GGT and Fe compared to adults. Marmosets housed strictly indoors showed increased ALT and GGT levels and decreased levels of total bilirubin and neutrophil count compared to marmosets housed with outdoor access. Pregnant marmosets showed increased ALP, total bilirubin, neutrophil count, monocyte count, and basophil count, and decreased levels of AST, ALT, cholesterol, Fe, and lymphocyte count compared to non-pregnant marmosets. Etonogestrel contracepted marmosets showed decreased P-LCR compared to females who were not contracepted. Updated reference intervals will aid researchers and veterinarians in identifying physiological and pathological changes, as well as improve the reproducibility of research in this species. [ABSTRACT FROM AUTHOR]

An abstract of the article "Interplay between AMA1 immunisation and parasite challenge leads to protection against malaria infection in rhesus macaques" that was presented at a conference titled "From Parasite to Prevention: Advances in the understanding of malaria" held in Edinburgh, Great Britain on October 20-22, 2010, is presented.

Objective To investigate the clinical and physiological effects of intravenous (IV) alfaxalone alone or in combination with buprenorphine, butorphanol or tramadol premedication in marmosets. Study design Prospective, randomized, blinded, crossover design. Animals Nine healthy marmosets (391 ± 48 g, 3.7 ± 2.2 years old). Methods Meloxicam 0.20 mg kg −1 subcutaneously, atropine 0.05 mg kg −1 intramuscularly (IM) and either buprenorphine 20 μg kg −1 IM (BUP-A), butorphanol 0.2 mg kg −1 IM (BUT-A), tramadol 1.5 mg kg −1 IM (TRA-A) or no additional drug (control) were administered to all marmosets as premedication. After 1 hour, anaesthesia was induced with 16 mg kg −1 alfaxalone IV. All animals received all protocols. The order of protocol allocation was randomized with a minimum 28 day wash-out period. During anaesthesia, respiratory and pulse rates, rectal temperature, haemoglobin oxygen saturation, arterial blood pressure, palpebral and pedal withdrawal reflexes and degree of muscle relaxation were assessed and recorded every 5 minutes. Quality of induction and recovery were assessed. Duration of induction, immobilization and recovery were recorded. Blood samples were analysed for aspartate aminotransferase, creatine kinase and lactate dehydrogenase concentrations. The protocols were compared using paired t tests, Wilcoxon's signed-rank test with Bonferroni's corrections and linear mixed effect models where appropriate. Results Out of nine animals, apnoea was noted in eight animals administered protocol BUP-A and two animals administered protocol BUT-A. With TRA-A and control protocols, apnoea was not observed. No other significant differences in any of the parameters were found; however, low arterial blood pressures and hypoxia occurred in TRA-A. Conclusions and clinical relevance Our study employing different premedications suggests that the previously published dose of 16 mg kg −1 alfaxalone is too high when used with premedication because we found a high incidence of complications including apnoea (BUP-A), hypotension and hypoxaemia (TRA-A). Appropriate monitoring and countermeasures are recommended. [ABSTRACT FROM AUTHOR]

Background: Coronavirus disease 2019 (COVID-19) patients initially develop respiratory symptoms, but they may also suffer from neurological symptoms. People with long-lasting effects after acute infections with severe respiratory syndrome coronavirus 2 (SARS-CoV-2), i.e., post-COVID syndrome or long COVID, may experience a variety of neurological manifestations. Although we do not fully understand how SARS-CoV-2 affects the brain, neuroinflammation likely plays a role. Methods: To investigate neuroinflammatory processes longitudinally after SARS-CoV-2 infection, four experimentally SARS-CoV-2 infected rhesus macaques were monitored for 7 weeks with 18-kDa translocator protein (TSPO) positron emission tomography (PET) using [18F]DPA714, together with computed tomography (CT). The baseline scan was compared to weekly PET–CTs obtained post-infection (pi). Brain tissue was collected following euthanasia (50 days pi) to correlate the PET signal with TSPO expression, and glial and endothelial cell markers. Expression of these markers was compared to brain tissue from uninfected animals of comparable age, allowing the examination of the contribution of these cells to the neuroinflammatory response following SARS-CoV-2 infection. Results: TSPO PET revealed an increased tracer uptake throughout the brain of all infected animals already from the first scan obtained post-infection (day 2), which increased to approximately twofold until day 30 pi. Postmortem immunohistochemical analysis of the hippocampus and pons showed TSPO expression in cells expressing ionized calcium-binding adaptor molecule 1 (IBA1), glial fibrillary acidic protein (GFAP), and collagen IV. In the hippocampus of SARS-CoV-2 infected animals the TSPO+ area and number of TSPO+ cells were significantly increased compared to control animals. This increase was not cell type specific, since both the number of IBA1+TSPO+ and GFAP+TSPO+ cells was increased, as well as the TSPO+ area within collagen IV+ blood vessels. Conclusions: This study manifests [18F]DPA714 as a powerful radiotracer to visualize SARS-CoV-2 induced neuroinflammation. The increased uptake of [18F]DPA714 over time implies an active neuroinflammatory response following SARS-CoV-2 infection. This inflammatory signal coincides with an increased number of TSPO expressing cells, including glial and endothelial cells, suggesting neuroinflammation and vascular dysregulation. These results demonstrate the long-term neuroinflammatory response following a mild SARS-CoV-2 infection, which potentially precedes long-lasting neurological symptoms. [ABSTRACT FROM AUTHOR]

Simple Summary: We explored the effect of age, gender, weight-for-height indices, sedation protocol, and housing conditions on the hematologic and serum biochemical values of captive rhesus and cynomolgus macaques. Several blood parameters demonstrated significant and clinically relevant changes in relation to the investigated variables. The results will provide veterinarians and researchers with important reference intervals for evaluating experimental results and health control from rhesus and cynomolgus macaques. Several physiological characteristics and housing conditions are known to affect hematologic and serum biochemical values in macaques. However, the studies that have been conducted either report values calculated based on a small number of animals, were designed specifically to document the effect of a particular condition on the normal range of hematologic and serum biochemical values, or used parametric assumptions to calculate hematologic and serum biochemical reference intervals. We conducted a retrospective longitudinal cohort study to estimate reference intervals for hematologic and serum biochemical values in clinically healthy macaques based on observed percentiles without parametric assumptions. Data were obtained as part of the Biomedical Primate Research Centre (Rijswijk, The Netherlands) health monitoring program between 2018 and 2021. In total, 4009 blood samples from 1475 macaques were analyzed with a maximum of one repeat per year per animal. Data were established by species, gender, age, weight-for-height indices, pregnancy, sedation protocol, and housing conditions. Most of the parameters profoundly affected just some hematologic and serum biochemical values. A significant glucose difference was observed between the ketamine and ketamine-medetomidine sedation protocols. The results emphasize the importance of establishing uniform experimental groups with validated animal husbandry and housing conditions to improve the reproducibility of the experiments. [ABSTRACT FROM AUTHOR]

In medicine and chemistry, immunoassays are often used to measure substance concentration. These tests use an S-shaped standard curve to map the observed optical responses to the underlying concentration. The enzyme-linked immunosorbent assay is one such test that is commonly used to measure antibody concentration in vaccine and infectious disease research. The enzyme-linked immunosorbent assay and other immunoassays usually involve a series of doubling or tripling dilutions of the test samples so that some of the diluted samples fall within the near-linear range in the center of the standard curve. The dilution that falls within or is nearest to the center of the near-linear range may then be selected for statistical analysis. This common practice of using one dilution does not fully use the information from multiple dilutions and reduces accuracy. We describe a recently proposed weighted-average estimation approach for analyzing multiple-dilution data (Cheung et al. 2015, Journal of Immunological Methods 417: 115-123), and we present the new wavemid command, which carries out the approach. We also present the new command midreshape, which processes raw data in text format exported from some microplate readers into analyzable data format. We use data from an experimental study of malaria vaccine candidates to demonstrate use of the two commands. [ABSTRACT FROM AUTHOR]

In medicine and chemistry, measurement of concentrations usually involves calibration that maps the observed responses to the underlying concentration using inversion of a standard curve. The Enzyme-linked ImmunoSorbent Assay (ELISA) is one example of such methods that is commonly used to measure antibody concentration. A problem in this and similar type of technology is that an accurate measurement is obtainable only if the observations fall within the optimal, near-linear range of the standard curve. It is common to conduct a series of doubling or tripling dilutions of the samples, so that at least some of the diluted samples are within the optimal range. A single dilution may then be selected for statistical analysis. This common practice does not fully utilize the data from multiple dilutions and reduces accuracy. We consider two weighted average estimators for fully utilizing the information from multiple dilutions. The first uses weights inversely proportional to the variances of the dilution-specific calibrated values; the second is a simplified form of the first. Simulation results demonstrated the superiority of this weighted estimation approach over the conventional approach of analyzing a single selected dilution. We apply the methods to an experimental study of malaria vaccine candidates. [ABSTRACT FROM AUTHOR]

Simple Summary: Indoor air quality is strongly associated with animal health and wellbeing. Therefore, animal enclosures must be consistently and sufficiently ventilated to provide for the health and well-being of animals and caretakers. Although there are several publications concerning assessments and effects of suboptimal air quality on rodents, no publications are available on group-housed non-human primates and the exposure of caretakers to inhalable dust and endotoxins. The indoor air quality of group-housed macaques at the Biomedical Primate Research Center (Rijswijk, the Netherlands) was assessed to identify possible problems regarding air circulation and the concentrations of inhalable dust, endotoxin, ammonia, fungi, temperature and relative humidity in the indoor environment. In addition, the exposure to inhalable dust and endotoxins of caretakers was evaluated. The observed values for these air quality parameters, measured at fixed locations in the animal enclosures, did not exceed the proposed human threshold limit values (TLV). However, caretakers were exposed to higher levels than the animals likely due to nature of their tasks. This study provides practical tools that can be used to improve the indoor air quality in group-housed macaques. Moreover, the results show that the exposure of caretakers to inhalable dust and endotoxins during daily work routines should be reduced. Indoor Air Quality (IAQ) is strongly associated with animal health and wellbeing. To identify possible problems of the indoor environment of macaques (Macaca spp.), we assessed the IAQ. The temperature (°C), relative humidity (%) and concentrations of inhalable dust (mg/m3), endotoxins (EU/m3), ammonia (ppm) and fungal aerosols were measured at stationary fixed locations in indoor enclosures of group-housed rhesus (Macaca mulatta) and cynomolgus macaques (Macaca fascicularis). In addition, the personal exposure of caretakers to inhalable dust and endotoxins was measured and evaluated. Furthermore, the air circulation was assessed with non-toxic smoke, and the number of times the macaques sneezed was recorded. The indoor temperature and relative humidity for both species were within comfortable ranges. The geometric mean (GM) ammonia, dust and endotoxin concentrations were 1.84 and 0.58 ppm, 0.07 and 0.07 mg/m3, and 24.8 and 6.44 EU/m3 in the rhesus and cynomolgus macaque units, respectively. The GM dust concentrations were significantly higher during the daytime than during the nighttime. Airborne fungi ranged between 425 and 1877 CFU/m3. Personal measurements on the caretakers showed GM dust and endotoxin concentrations of 4.2 mg/m3 and 439.0 EU/m3, respectively. The number of sneezes and the IAQ parameters were not correlated. The smoke test revealed a suboptimal air flow pattern. Although the dust, endotoxins and ammonia were revealed to be within accepted human threshold limit values (TLV), caretakers were exposed to dust and endotoxin levels exceeding existing occupational reference values. [ABSTRACT FROM AUTHOR]

Common marmosets (Callithrix jacchus) demonstrate variations in reproductive output, not only in terms of total reproductive output during a lifetime but also in litter size per parturition. The present study explores factors, such as parents' litter size, parturition number, maternal body weight at conception and maternal age, which may account for this variation. A retrospective analysis of clinical records of a captive breeding colony was conducted over a 9‐year period yielding reproductive summaries of 26 dams and 22 sires producing a total of 115 litters. Dams born from litters of  ≤2 (N = 20) more often produced litters of ≤2, whereas dams born from litters of >2 (N = 6) more often produced litters of >2 (p < 0.05). The dams' maternal body weight at the time of conception had also a significant effect on subsequent litter size. In addition, the chance of triplets was higher after the second parturition. Maternal age, interbirth interval, and season of birth had no effect on litter size. Factors relating to the sire had a negligible effect on the size of the litter. Multivariate statistical modeling revealed that the dams' original litter size, maternal bodyweight at conception and parturition number are determining factors for the number of babies per litter. This study identified factors determining marmoset litter size, some of which (maternal litter size) are novel to this study and were not reported previously. Further exploration of the potential role of maternal litter size as a determinant of the litter sizes produced by marmoset breeders is warranted. Dams born from a twin litter are more likely to give birth to twin litters and dams born from triplet litters are more likely to produce triplets. Parturition number and maternal body weight at time of conception significantly influenced the produced litter size. The influence of the sire on the litter size is negligible. [ABSTRACT FROM AUTHOR]

Background: Substantial evidence indicates that cytophilic IgG responses to Plasmodium falciparum merozoite antigens play a role in protection from malaria. The specific targets mediating immunity remain unclear. Evaluating antibody responses in infants naturally-exposed to malaria will allow to better understand the establishment of anti-malarial immunity and to contribute to a vaccine development by identifying the most appropriate merozoite candidate antigens. Methods: The study was based on parasitological and clinical active follow-up of infants from birth to 18 months of age conducted in the Tori Bossito area of southern Benin. For 399 infants, plasma levels of cytophilic IgG antibodies with specificity for five asexual stage malaria vaccine candidate antigens were determined by ELISA in infants' peripheral blood at 6, 9, 12 and 15 months of age. Multivariate mixed logistic model was used to investigate the association between antibody levels and anti-malarial protection in the trimester following the IgG quantification. Moreover, the concentrations of merozoite antigen-specific IgG were compared between a group of infants apparently able to control asymptomatic malaria infection (CAIG) and a group of infants with no control of malaria infection (Control group (NCIG)). Protective effect of antibodies was also assessed after 15 months of malaria exposure with a Cox regression model adjusted on environmental risk. Results: Cytophilic IgG responses to AMA1, MSP1, MSP2-3D7, MSP2-FC27, MSP3 and GLURP R2 were associated with increasing malarial infection risk in univariate analysis. The multivariate mixed model showed that IgG1 and IgG3 to AMA1 were associated with an increased risk of malarial infection. However infants from CAIG (n = 53) had significantly higher AMA1-, MSP2-FC27-, MSP3-specific IgG1 and AMA1-, MSP1-, MSP2-FC27-, MSP3 and GLURP-R2-specific IgG3 than those from NCIG (n = 183). The latter IgG responses were not associated with protection against clinical malaria in the whole cohort when protective effect is assessed after 15 months of malaria exposition. Conclusion: In this cohort, merozoite antigen-specific cytophilic IgG levels represent a marker of malaria exposure in infants from 6 to 18 months of age. However, infants with resolution of asymptomatic infection (CAIG) seem to have acquired naturally immunity against P. falciparum. This observation is encouraging in the context of the development of multitarget P. falciparum vaccines. [ABSTRACT FROM AUTHOR]

Abstract A major challenge in influenza research is the selection of an appropriate animal model that accurately reflects the disease and the protective immune response observed in humans. A workshop organised by the EDUFLUVAC consortium, a European Union funded project coordinated by the European Vaccine Initiative, brought together experts from the influenza vaccine community with the aim to discuss the current knowledge and future perspectives for testing broadly reactive influenza vaccines in animal models. The programme included a diversity of models from well-established and publicly accepted models to cutting edge, newly developed animal models as well as ex-vivo approaches and human models. The audience concluded that different vaccine approaches may require evaluation in different animal models, depending on the type of immune response induced by the vaccine. Safety is the main concern for transition to clinical development and influenza vaccine associated enhanced disease was specifically emphasised. An efficient animal model to evaluate this aspect of safety still needs to be identified. Working with animal models requires ethical compliance and consideration of the 3R principles. Development of alternative approaches such as ex-vivo techniques is progressing but is still at an early stage and these methods are not yet suitable for broader application for vaccine evaluation. The human challenge is the ultimate model to assess influenza vaccines. However this model is expensive and not largely applicable. The currently used pre-clinical models are not yet specifically focused on studying unique aspects of a universal influenza vaccine. Further collaboration, communication and effective networking are needed for success in establishment of harmonised and standardised pre-clinical models for evaluation of new influenza vaccines. This report does not provide a complete review of the field but discusses the data presented by the speakers and discussion points raised during the meeting. [ABSTRACT FROM AUTHOR]

Our human model of nasal colonization and eradication of S. aureus is limited by safety issues. As rhesus macaques are closely related to humans and natural hosts for S. aureus, we developed an experimental decolonization and inoculation protocol in these animals. Animals were screened for nasal carriage of S. aureus and 20 carriers were selected. Decolonization was attempted using nasal mupirocin (10 animals) or mupirocin plus trimethoprim/sulfadiazine intramuscularly (10 animals) both once daily for 5 days, and checked by follow-up cultures for 10 weeks. Intranasal inoculation was performed with S. aureus strain 8325–4 in culture-negative animals. 11/20 animals, of which 5 received mupirocin and 6 the combination treatment, became culture-negative for S. aureus for 10 weeks and these 11 animals were subsequently inoculated. Swabs were taken once a week for 5 weeks to test for the presence of the inoculated strain. In 3 animals, strain 8325–4 was cultured from the nose 1 week after inoculation, indicating short-term survival of this strain only, a finding similar to that previously found in our human model. These data demonstrate that rhesus macaques may constitute a relevant animal model to perform S. aureus eradication and inoculation studies with relatively limited invasive handling of the animals. [ABSTRACT FROM AUTHOR]

Introduction: Plasmodium falciparum induced antibodies are key components of anti-malarial immunity in malaria endemic areas, but their antigen targets can be polymorphic. Induction of a high proportion of strain-specific antibodies will limit the recognition of a broad diversity of parasite strains by these responses. There are indications that circulating parasite diversity varies with malaria transmission intensity, and this may affect the specificity of elicited anti-malarial antibodies. This study therefore assessed the effect of varying malaria transmission patterns on the specificity of elicited antibody responses and to identify possible antibody correlates of naturally acquired immunity to malaria in children in an area of Ghana with seasonal malaria transmission. Methods: This retrospective study utilized plasma samples collected longitudinally at six time points from children aged one to five years. Multiplex assays were used to measure antibody levels against four P. falciparum AMA 1 variants (from the 3D7, FVO, HB3 and CAMP parasite strains) and the 3D7 variant of the EBA 175 region II antigen and the levels compared between symptomatic and asymptomatic children. The relative proportions of cross-reactive and strain-specific antibodies against the four AMA 1 variants per sampling time point were assessed by Bland-Altman plots. The levels of antibodies against allelic AMA1 variants, measured by singleplex and multiplex luminex assays, were also compared. Results: The data show that increased transmission intensity is associated with higher levels of cross-reactive antibody responses, most likely a result of a greater proportion of multiple parasite clone infections during the high transmission period. Anti-AMA1 antibodies were however associated with a history of infection rather than protection in this age group. Conclusion: The data contribute to understanding the underlying mechanism of the acquisition of strain-transcending antibody immunity following repeated exposure to diverse parasite strains. [ABSTRACT FROM AUTHOR]

The development of broadly reactive influenza vaccines raises the need to identify the most appropriate immunoassays that can be used for the evaluation of so-called universal influenza vaccines and to explore a path towards the standardisation of such assays. More than fifty experts from the global influenza vaccine research and development field met to initiate such discussion at a workshop co-organised by the EDUFLUVAC consortium, a European Union funded project coordinated by the European Vaccine Initiative, and the National Institutes of Health/National Institute of Allergy and Infectious Diseases, USA. The workshop audience agreed that it was not possible to establish a single immunoassay for "universal" influenza vaccines because the current approaches differ in the vaccines' nature and immunogenicity properties. Therefore, different scientific rationales for the immunoassay selection are required. To avoid dilution of efforts, the choice of the primary evaluation criteria (eg serological assays or T-cell assays) should drive the effort of harmonisation. However, at an early phase of clinical development, more efforts on exploratory assessments should be undertaken to better define the immune profile in response to immunisation with new vaccines. The workshop concluded that each laboratory should aim towards validation of the appropriate immunoassays used during the entire process of vaccine development from antigen discovery up to establishment of correlates of protection, including the different steps of quality control (eg potency assays), animal studies and human clinical development. Standardisation of the immunoassays is the ultimate goal, and there is a long way to go. [ABSTRACT FROM AUTHOR]

Whole malaria sporozoite vaccine regimens are promising new strategies, and some candidates have demonstrated high rates of durable clinical protection associated with memory T cell responses. Little is known about the anatomical distribution of memory T cells following whole sporozoite vaccines, and immunization of nonhuman primates can be used as a relevant model for humans. We conducted a chemoprophylaxis with sporozoite (CPS) immunization in P. knowlesi rhesus monkeys and challenged via mosquito bites. Half of CPS immunized animals developed complete protection, with a marked delay in parasitemia demonstrated in the other half. Antibody responses to whole sporozoites, CSP, and AMA1, but not CelTOS were detected. Peripheral blood T cell responses to whole sporozoites, but not CSP and AMA1 peptides were observed. Unlike peripheral blood, there was a high frequency of sporozoite-specific memory T cells observed in the liver and bone marrow. Interestingly, sporozoite-specific CD4+ and CD8+ memory T cells in the liver highly expressed chemokine receptors CCR5 and CXCR6, both of which are known for liver sinusoid homing. The majority of liver sporozoite-specific memory T cells expressed CD69, a phenotypic marker of tissue-resident memory (TRM) cells, which are well positioned to rapidly control liver-stage infection. Vaccine strategies that aim to elicit large number of liver TRM cells may efficiently increase the efficacy and durability of response against pre-erythrocytic parasites. [ABSTRACT FROM AUTHOR]

Plasmodium falciparum apical membrane antigen 1 (PfAMA1) is a leading asexual blood stage vaccine candidate for malaria. In preparation for clinical trials, three Diversity Covering (DiCo) PfAMA1 ectodomain proteins, designed to overcome the intrinsic polymorphism that is present in PfAMA1, were produced under Good Manufacturing Practice (GMP) in Pichia pastoris. Using identical methodology, the 3 strains were cultivated in 70-L scale fed-batch fermentations and PfAMA1-DiCos were purified by two chromatography steps, an ultrafiltration/diafiltration procedure and size exclusion chromatography, resulting in highly pure (>95%) PfAMA1-DiCo1, PfAMA1 DiCo2 and PfAMA1 DiCo3, with final yields of 1.8, 1.9 and 1.3 gram, respectively. N-terminal determinations showed that approximately 50% of each of the proteins lost 12 residues from their N-terminus, in accordance with SDS-PAGE (2 main bands) and MS-data. Under reducing conditions a site of limited proteolytic cleavage within a disulphide bonded region became evident. The three proteins quantitatively bound to the mAb 4G2 that recognizes a conformational epitope, suggesting proper folding of the proteins. The lyophilized Drug Product (1:1:1 mixture of PfAMA1-DiCo1, DiCo2, DiCo3) fulfilled all pre-set release criteria (appearance, dissolution rate, identity, purity, protein content, moisture content, sub-visible particles, immuno-potency (after reconstitution with adjuvant), abnormal toxicity, sterility and endotoxin), was stable in accelerated and real-time stability studies at -20°C for over 24 months. When formulated with adjuvants selected for clinical phase I evaluation, the Drug Product did not show adverse effect in a repeated-dose toxicity study in rabbits. The Drug Product has entered a phase Ia/Ib clinical trial. [ABSTRACT FROM AUTHOR]

Antibodies that impede the invasion of Plasmodium falciparum ( P. falciparum ) merozoites into erythrocytes play a critical role in anti-malarial immunity. The Growth Inhibition Assay (GIA) is an in vitro measure of the functional capacity of such antibodies to limit erythrocyte invasion and/or parasite growth. Up to now, it is unclear whether growth-inhibitory activity correlates with protection from clinical disease and there are inconsistent results from studies performed with GIA. Studies that have focused on the relationship between IgGs and their in vitro parasite Growth Inhibition Activity (GIAc) in infants aged less than two years old are rare. Here, we used clinical and parasitological data to precisely define symptomatic or asymptomatic infection with P. falciparum in groups of infants followed-up actively for 18 months post-natally. We quantified the levels of IgG1 and IgG3 directed to a panel of candidate P. falciparum vaccine antigens (AMA-1, MSP1, 2, 3 and GLURP) using ELISA and the functional activity of IgG was quantified using GIA. Data were then correlated with individuals' infection status. At 18 months of age, infants harbouring infections at the time of blood sampling had an average 19% less GIAc than those not infected ( p = 0.004, multivariate linear regression ). GIAc decreased from 12 to 18 months of age ( p = 0.003, Wilcoxon matched pairs test ). Antibody levels quantified at 18 months in infants were strongly correlated with their exposure to malarial infection, however GIAc was not correlated with malaria infectious status (asymptomatic and symptomatic groups). In conclusion, both infection status at blood draw and age influence parasite growth inhibition mediated by IgG in the GIA. Both factors must be taken into account when correlations between GIAc and anti-malarial protection or vaccine efficacy have to be made. [ABSTRACT FROM AUTHOR]

Influenza virus infection of nonhuman primates is a well-established animal model for studying pathogenesis and for evaluating prophylactic and therapeutic intervention strategies. However, usually a standard dose is used for the infection, and there is no information on the relation between challenge dose and virus replication or the induction of immune responses. Such information is also very scarce for humans and largely confined to evaluation of attenuated virus strains. Here, we have compared the effect of a commonly used dose (4×106 50% tissue culture infective doses) versus a 100-fold-higher dose, administered by intrabronchial installation, to two groups of 6 cynomolgus macaques. Animals infected with the high virus dose showed more fever and had higher peak levels of gamma interferon in the blood. However, virus replication in the trachea was not significantly different between the groups, although in 2 out of 6 animals from the high-dose group it was present at higher levels and for a longer duration. The virus-specific antibody response was not significantly different between the groups. However, antibody enzyme-linked immunosorbent assay, virus neutralization, and hemagglutination inhibition antibody titers correlated with cumulative virus production in the trachea. In conclusion, using influenza virus infection in cynomolgus macaques as a model, we demonstrated a relationship between the level of virus production upon infection and induction of functional antibody responses against the virus. [ABSTRACT FROM AUTHOR]

Prospects for malaria eradication will be greatly enhanced by an effective vaccine, but parasite genetic diversity poses a major impediment to malaria vaccine efficacy. In recent pre-clinical and field trials, vaccines based on polymorphic Plasmodium falciparum antigens have shown efficacy only against homologous strains, raising the specter of allele-specific immunity such as that which plagues vaccines against influenza and HIV. The most advanced malaria vaccine, RTS,S, targets relatively conserved epitopes on the P. falciparum circumsporozoite protein. After more than 40 years of development and testing, RTS,S, has shown significant but modest efficacy against clinical malaria in phase 2 and 3 trials. Ongoing phase 2 studies of an irradiated sporozoite vaccine will ascertain whether the full protection against homologous experimental malaria challenge conferred by high doses of a whole organism vaccine can provide protection against diverse strains in the field. Here we review and evaluate approaches being taken to design broadly cross-protective malaria vaccines. [ABSTRACT FROM AUTHOR]

The close immunological and physiological resemblance with humans makes non-human primates a valuable model for studying influenza virus pathogenesis and immunity and vaccine efficacy against infection. Although both cynomolgus and rhesus macaques are frequently used in influenza virus research, a direct comparison of susceptibility to infection and disease has not yet been performed. In the current study a head-to-head comparison was made between these species, by using a recently described swine-origin pandemic H1N1 strain, A/Mexico/InDRE4487/2009. In comparison to rhesus macaques, cynomolgus macaques developed significantly higher levels of virus replication in the upper airways and in the lungs, involving both peak level and duration of virus production, as well as higher increases in body temperature. In contrast, clinical symptoms, including respiratory distress, were more easily observed in rhesus macaques. Expression of sialyl-α-2,6-Gal saccharides, the main receptor for human influenza A viruses, was 50 to 73 times more abundant in trachea and bronchus of cynomolgus macaques relative to rhesus macaques. The study also shows that common marmosets, a New World non-human primate species, are susceptible to infection with pandemic H1N1. The study results favor the cynomolgus macaque as model for pandemic H1N1 influenza virus research because of the more uniform and high levels of virus replication, as well as temperature increases, which may be due to a more abundant expression of the main human influenza virus receptor in the trachea and bronchi. [ABSTRACT FROM AUTHOR]

Infection with Plasmodium knowlesi, a zoonotic primate malaria, is a growing human health problem in Southeast Asia. P. knowlesi is being used in malaria vaccine studies, and a number of proteins are being considered as candidate malaria vaccine antigens, including the Apical Membrane Antigen 1 (AMA1). In order to determine genetic diversity of the ama1 gene and to identify epitopes of AMA1 under strongest immune selection, the ama1 gene of 52 P. knowlesi isolates derived from human infections was sequenced. Sequence analysis of isolates from two geographically isolated regions in Sarawak showed that polymorphism in the protein is low compared to that of AMA1 of the major human malaria parasites, P. falciparum and P. vivax. Although the number of haplotypes was 27, the frequency of mutations at the majority of the polymorphic positions was low, and only six positions had a variance frequency higher than 10%. Only two positions had more than one alternative amino acid. Interestingly, three of the high-frequency polymorphic sites correspond to invariant sites in PfAMA1 or PvAMA1. Statistically significant differences in the quantity of three of the six high frequency mutations were observed between the two regions. These analyses suggest that the pkama1 gene is not under balancing selection, as observed for pfama1 and pvama1, and that the PkAMA1 protein is not a primary target for protective humoral immune responses in their reservoir macaque hosts, unlike PfAMA1 and PvAMA1 in humans. The low level of polymorphism justifies the development of a single allele PkAMA1-based vaccine. [ABSTRACT FROM AUTHOR]

Background: Handling of common marmoset (Callithrix jacchus) usually requires chemical restraint. Ketamine has been associated with muscle damage in primates, while common marmosets, compared to other primates, additionally display an exceptional high sensitivity to ketamine-associated side-effects. Notably, muscle twitching movements of limbs and hands, and a marked increase in salivation are observed. We investigated two alternative intramuscular (i.m.) immobilisation protocols against ketamine (50 mg/kg; protocol 1) in a double-blind randomised crossover study in ten healthy adult common marmosets for use as a safe reliable, short-term immobilisation and sedation. These protocols comprised: alphaxalone (12 mg/kg; protocol 2) and 25 mg/kg ketamine combined with 0.50 mg/kg medetomidine (reversal with 2.5 mg/kg atipamezole; protocol 3A). Following completion and unblinding, the project was extended with an additional protocol (3B), comprising 25 mg/kg ketamine combined with 0.05 mg/kg medetomidine (reversal with 0.25 mg/kg atipamezole, twice with 35 min interval). Results: All protocols in this study provided rapid onset (induction times <5 min) of immobilisation and sedation. Duration of immobilisation was 31.23 ± 22.39 min, 53.72 ± 13.08 min, 19.73 ± 5.74 min, and 22.78 ± 22.37 min for protocol 1, 2, 3A, and 3B, respectively. Recovery times were 135.84 ± 39.19 min, 55.79 ± 11.02 min, 405.46 ± 29.81 min, and 291.91 ± 80.34 min, respectively. Regarding the quality, and reliability (judged by pedal withdrawal reflex, palpebral reflex and muscle tension) of all protocols, protocol 2 was the most optimal. Monitored vital parameters were within clinically acceptable limits during all protocols and there were no fatalities. Indication of muscle damage as assessed by AST, LDH and CK values was most prominent elevated in protocol 1, 3A, and 3B. Conclusions: We conclude that intramuscular administration of 12 mg/kg alphaxalone to common marmosets is preferred over other protocols studied. Protocol 2 resulted in at least comparable immobilisation quality with acceptable and less frequent side effects and superior recovery quality. In all protocols, supportive therapy, such as external heat support, remains mandatory. Notably, an unacceptable long recovery period in both ketamine/ medetomidine protocols (subsequently reversed with atipamezole) was observed, showing that α-2 adrenoreceptor agonists in the used dose and dosing regime is not the first choice for sedation in common marmosets in a standard research setting. [ABSTRACT FROM AUTHOR]

Volunteers immunized under chloroquine chemoprophylaxis with Plasmodium falciparum sporozoites (CPS) develop complete, long-lasting protection against homologous sporozoite challenge. Chloroquine affects neither sporozoites nor liver-stages, but kills only asexual forms in erythrocytes once released from the liver into the circulation. Consequently, CPS immunization exposes the host to antigens from both preerythrocytic and blood stages, and induced immunity might target either of these stages. We therefore explored the life cycle stage specificity of CPS-induced protection. Twenty-five malaria-naïve volunteers were enrolled in a clinical trial, 15 of whom received CPS immunization. Five immunized subjects and five controls received a sporozoite challenge by mosquito bites, whereas nine immunized and five control subjects received an i.v. challenge with P. falciparum-infected erythrocytes. The latter approach completely bypasses preerythrocytic stages, enabling a direct comparison of protection against either life cycle stage. CPS-immunized subjects (13 of 14) developed anticircumsporozoite antibodies, whereas only one volunteer generated minimal titers against typical blood-stage antigens. IgG from CPS-immunized volunteers did not inhibit asexual blood-stage growth in vitro. All CPS-immunized subjects (5 of 5) were protected against sporozoite challenge. In contrast, nine of nine CPS-immunized subjects developed parasitemia after blood-stage challenge, with identical prepatent periods and blood-stage multiplication rates compared with controls. Intravenously challenged CPS- immunized subjects showed earlier fever and increased plasma concentrations of inflammatory markers D-dimer, IFN -?, and monokine induced by IFN-γ? than i.v. challenged controls. The complete lack of protection against blood-stage challenge indicates that CPS-induced protection is mediated by immunity against preerythrocytic stages. However, evidence is presented for immune recognition of P. falciparum-infected erythrocytes, suggesting memory responses unable to generate functional immunity. [ABSTRACT FROM AUTHOR]

Background: Malaria transmission is highly heterogeneous in most settings, resulting in the formation of recognizable malaria hotspots. Targeting these hotspots might represent a highly efficacious way of controlling or eliminating malaria if the hotspots fuel malaria transmission to the wider community. Methods/design: Hotspots of malaria will be determined based on spatial patterns in age-adjusted prevalence and density of antibodies against malaria antigens apical membrane antigen-1 and merozoite surface protein-1. The community effect of interventions targeted at these hotspots will be determined. The intervention will comprise larviciding, focal screening and treatment of the human population, distribution of long-lasting insecticide-treated nets and indoor residual spraying. The impact of the intervention will be determined inside and up to 500 m outside the targeted hotspots by PCR-based parasite prevalence in cross-sectional surveys, malaria morbidity by passive case detection in selected facilities and entomological monitoring of larval and adult Anopheles populations. Discussion: This study aims to provide direct evidence for a community effect of hotspot-targeted interventions. The trial is powered to detect large effects on malaria transmission in the context of ongoing malaria interventions. Follow-up studies will be needed to determine the effect of individual components of the interventions and the cost-effectiveness of a hotspot-targeted approach, where savings made by reducing the number of compounds that need to receive interventions should outweigh the costs of hotspot-detection. [ABSTRACT FROM AUTHOR]

Abstract: Transfusion transmitted malaria (TTM) in non-endemic countries is reduced by questioning blood donors and screening of donated blood. Conventional screening is performed by Indirect Fluorescence Antibody Test (IFAT). This method is manual and difficult to standardize. Here we study the diagnostic performance of a multiplex assay for detection of antibodies against Plasmodium falciparum in donor blood using IFAT as a comparator. A multiplex assay (MPA) containing the antigens GLURP-R0, GLURP-R2, MSP3, MSP1 hybrid and AMA1 was constructed using xMAP® technology. A discrimination index for exposure to P. falciparum malaria was calculated by comparing travelers with clinical malaria (n=52) and non-exposed blood donors (n=119). The index was evaluated on blood donors with suspected malaria exposure (n=249) and compared to the diagnostic performance of IFAT. At a specificity of 95.8 %, the MPA discrimination index exhibited a diagnostic sensitivity of 90.4 % in travelers hospitalized with malaria. Percent agreement with IFAT was 92.3 %. Screening plasma from blood donors with suspected malaria exposure, we found 4.8 % to be positive by IFAT and 5.2 % by MPA with an agreement of 93.2 %. The calculated index from the MPA exhibits similar diagnostic performance as IFAT for detection of P. falciparum malaria. Combining the antibody response against multiple antigens in a discrimination index increased the sensitivity of the MPA and reduced the readout to a single value. [Copyright &y& Elsevier]

Polymorphic parasite antigens are known targets of protective immunity to malaria, but this antigenic variation poses challenges to vaccine development. A synthetic MSP-1 Block 2 construct, based on all polymorphic variants found in natural Plasmodium falciparum isolates has been designed, combined with the relatively conserved Block 1 sequence of MSP-1 and expressed in E.coli. The MSP-1 Hybrid antigen has been produced with high yield by fed-batch fermentation and purified without the aid of affinity tags resulting in a pure and extremely thermostable antigen preparation. MSP-1 hybrid is immunogenic in experimental animals using adjuvants suitable for human use, eliciting antibodies against epitopes from all three Block 2 serotypes. Human serum antibodies from Africans naturally exposed to malaria reacted to the MSP-1 hybrid as strongly as, or better than the same serum reactivities to individual MSP-1 Block 2 antigens, and these antibody responses showed clear associations with reduced incidence of malaria episodes. The MSP-1 hybrid is designed to induce a protective antibody response to the highly polymorphic Block 2 region of MSP-1, enhancing the repertoire of MSP-1 Block 2 antibody responses found among immune and semi-immune individuals in malaria endemic areas. The target population for such a vaccine is young children and vulnerable adults, to accelerate the acquisition of a full range of malaria protective antibodies against this polymorphic parasite antigen. [ABSTRACT FROM AUTHOR]

Background: Malaria caused by Plasmodium falciparum remains a major cause of death in sub-Saharan Africa. Immunity against symptoms of malaria requires repeated exposure, suggesting either that the parasite is poorly immunogenic or that the development of effective immune responses to malaria may be impaired. Methods: We carried out two age-stratified cross-sectional surveys of anti-malarial humoral immune responses in a Gambian village where P. falciparum malaria transmission is low and sporadic. Circulating antibodies and memory B cells (MBC) to four malarial antigens were measured using ELISA and cultured B cell ELISpot. Findings and Conclusions: The proportion of individuals with malaria-specific MBC and antibodies, and the average number of antigens recognised by each individual, increased with age but the magnitude of these responses did not. Malaria-specific antibody levels did not correlate with either the prevalence or median number of MBC, indicating that these two assays are measuring different aspects of the humoral immune response. Among those with immunological evidence of malaria exposure (defined as a positive response to at least one malarial antigen either by ELISA or ELISPOT), the median number of malaria-specific MBC was similar to median numbers of diphtheria-specific MBC, suggesting that the circulating memory cell pool for malaria antigens is of similar size to that for other antigens. [ABSTRACT FROM AUTHOR]

Background: Apical membrane antigen 1 (AMA1) is a leading candidate vaccine antigen against blood-stage malaria, although to date numerous clinical trials using mainly protein-in-adjuvant vaccines have shown limited success. Here we describe the pre-clinical development and optimization of recombinant human and simian adenoviral (AdHu5 and ChAd63) and orthopoxviral (MVA) vectors encoding transgene inserts for Plasmodium falciparum AMA1 (PfAMA1). Methodology/Principal Findings: AdHu5-MVA prime-boost vaccination in mice and rabbits using these vectors encoding the 3D7 allele of PfAMA1 induced cellular immune responses as well as high-titer antibodies that showed growth inhibitory activity (GIA) against the homologous but not heterologous parasite strains. In an effort to overcome the issues of PfAMA1 antigenic polymorphism and pre-existing immunity to AdHu5, a simian adenoviral (ChAd63) vector and MVA encoding two alleles of PfAMA1 were developed. This antigen, composed of the 3D7 and FVO alleles of PfAMA1 fused in tandem and with expression driven by a single promoter, was optimized for antigen secretion and transmembrane expression. These bi-allelic PfAMA1 vaccines, when administered to mice and rabbits, demonstrated comparable immunogenicity to the mono-allelic vaccines and purified serum IgG now showed GIA against the two divergent strains of P. falciparum encoded in the vaccine. CD8+ and CD4+ T cell responses against epitopes that were both common and unique to the two alleles of PfAMA1 were also measured in mice. Conclusions/Significance: Optimized transgene inserts encoding two divergent alleles of the same antigen can be successfully inserted into adeno- and pox-viral vaccine vectors. Adenovirus-MVA immunization leads to the induction of T cell responses common to both alleles, as well as functional antibody responses that are effective against both of the encoded strains of P. falciparum in vitro. These data support the further clinical development of these vaccine candidates in Phase I/IIa clinical trials. [ABSTRACT FROM AUTHOR]

Background: Antibodies to key Plasmodium falciparum surface antigens have been shown to be important effectors that mediate clinical immunity to malaria. The cross-strain fraction of anti-malarial antibodies may however be required to achieve strain-transcending immunity. Such antibody responses against Plasmodium falciparum apical membrane antigen 1 (PfAMA1), a vaccine target molecule that is expressed in both liver and blood stages of the parasite, can be elicited through immunization with a mixture of allelic variants of the parasite molecule. Cross-strain antibodies are most likely elicited against epitopes that are shared by the allelic antigens in the vaccine cocktail. Methods: A standard competition ELISA was used to address whether the antibody response can be further focused on shared epitopes by exclusively boosting these common determinants through immunization of rabbits with different PfAMA1 alleles in sequence. The in vitro parasite growth inhibition assay was used to further evaluate the functional effects of the broadened antibody response that is characteristic of multi-allele vaccine strategies. Results: A mixed antigen immunization protocol elicited humoral responses that were functionally similar to those elicited by a sequential immunization protocol (p > 0.05). Sequential exposure to the different PfAMA1 allelic variants induced immunological recall of responses to previous alleles and yielded functional cross-strain antibodies that would be capable of optimal growth inhibition of variant parasites at high enough concentrations. Conclusions: These findings may have implications for the current understanding of the natural acquisition of clinical immunity to malaria as well as for rational vaccine design. [ABSTRACT FROM AUTHOR]

There is increasing interest in multi-allele vaccines to overcome strain-specificity against polymorphic vaccine targets such as Apical Membrane Antigen 1 (AMA1). These have been shown to induce broad inhibitory antibodies in vitro and formed the basis for the design of three Diversity-Covering (DiCo) proteins with similar immunological effects. The antibodies produced are to epitopes that are shared between vaccine alleles and theoretically, increasing the number of component AMA1 alleles is expected to broaden the antibody response. A plateau effect could however impose a limit on the number of alleles needed to achieve the broadest specificity. Moreover, production cost and the vaccine formulation process would limit the number of component alleles. In this paper, we compare rabbit antibody responses elicited with multi-allele vaccines incorporating seven (three DiCos and four natural AMA1 alleles) and three (DiCo mix) antigens for gains in broadened specificity. We also investigate the effect of three adjuvant platforms on antigen specificity and antibody functionality. Our data confirms a broadened response after immunisation with DiCo mix in all three adjuvants. Higher antibody titres were elicited with either CoVaccine HTTM or Montanide ISA 51, resulting in similar in vitro inhibition (65-82%) of five out of six culture-adapted P. falciparum strains. The antigen binding specificities of elicited antibodies were also similar and independent of the adjuvant used or the number of vaccine component alleles. Thus neither the four extra antigens nor adjuvant had any observable benefits with respect to specificity broadening, although adjuvant choice influenced the absolute antibody levels and thus the extent of parasite inhibition. Our data confirms the feasibility and potential of multi-allele PfAMA1 formulations, and highlights the need for adjuvants with improved antibody potentiation properties for AMA1-based vaccines. [ABSTRACT FROM AUTHOR]

In the absence of treatment, most HIV-1-infected humans develop AIDS. However, a minority are long-term nonprogressors, and resistance is associated with the presence of particular HLA-B*27/ B*57 molecules. In contrast, most HIV-1-infected chimpanzees do not contract AIDS. In comparison with humans, chimpanzees experienced an ancient selective sweep affecting the MHC class I repertoire. We have determined the peptide-binding properties of frequent chimpanzee MHC class I molecules, and show that, like HLA-B*27IB*57, they target similar conserved areas of HIV1/SlVcpz. In addition, many animals appear to possess multiple molecules targeting various conserved areas of the HlV-1/SlVcpz Gag protein, a quantitative aspect of the immune response that may further minimize the chance of viral escape. The functional characteristics of the contemporary chimpanzee MHC repertoire suggest that the selective sweep was caused by a lentiviral pandemic. [ABSTRACT FROM AUTHOR]

Apical Membrane Antigen 1 (AMA1), a merozoite protein essential for red cell invasion, is a candidate malaria vaccine component. Immune responses to AMA1 can protect in experimental animal models and antibodies isolated from AMA1- vaccinated or malaria-exposed humans can inhibit parasite multiplication in vitro. The parasite is haploid in the vertebrate host and the genome contains a single copy of AMA1, yet on a population basis a number of AMA1 molecular surface residues are polymorphic, a property thought to be primarily as a result of selective immune pressure. After immunisation with AMA1, antibodies more effectively inhibit strains carrying homologous AMA1 genes, suggesting that polymorphism may compromise vaccine efficacy. Here, we analyse induction of broad strain inhibitory antibodies with a multi-allele Plasmodium falciparum AMA1 (PfAMA1) vaccine, and determine the relative importance of cross-reactive and strain-specific IgG fractions by competition ELISA and in vitro parasite growth inhibition assays. Immunisation of rabbits with a PfAMA1 allele mixture yielded an increased proportion of antibodies to epitopes common to all vaccine alleles, compared to single allele immunisation. Competition ELISA with the anti-PfAMA1 antibody fraction that is cross-reactive between FVO and 3D7 AMA1 alleles showed that over 80% of these common antibodies were shared with other PfAMA1 alleles. Furthermore, growth inhibition assays revealed that for any PfAMA1 allele (FVO or 3D7), the cross-reactive fraction alone, on basis of weight, had the same functional capacity on homologous parasites as the total affinity-purified IgGs (cross-reactive+strain-specific). By contrast, the strain-specific IgG fraction of either PfAMA1 allele showed slightly less inhibition of red cell invasion by homologous strains. Thus multi-allele immunisation relatively increases the levels of antibodies to common allele epitopes. This explains the broadened cross inhibition of diverse malaria parasites, and suggests multi-allele approaches warrant further clinical investigation. [ABSTRACT FROM AUTHOR]

Background: Antibodies, particularly cytophilic IgG subclasses, with specificity for asexual blood stage antigens of Plasmodium falciparum, are thought to play an important role in acquired immunity to malaria. Evaluating such responses in longitudinal sero-epidemiological field studies, allied to increasing knowledge of the immunological mechanisms associated with anti-malarial protection, will help in the development of malaria vaccines. Methods and Findings: We conducted a 1-year follow-up study of 305 Senegalese children and identified those resistant or susceptible to malaria. In retrospective analyses we then compared post-follow-up IgG responses to six asexual-stage candidate malaria vaccine antigens in groups of individuals with clearly defined clinical and parasitological histories of infection with P. falciparum. In age-adjusted analyses, children resistant to malaria as well as to high-density parasitemia, had significantly higher IgG1 responses to GLURP and IgG3 responses to MSP2 than their susceptible counterparts. Among those resistant to malaria, high anti-MSP1 IgG1 levels were associated with protection against high-density parasitemia. To assess functional attributes, we used an in vitro parasite growth inhibition assay with purified IgG. Samples from individuals with high levels of IgG directed to MSP1, MSP2 and AMA1 gave the strongest parasite growth inhibition, but a marked agerelated decline was observed in these effects. Conclusion: Our data are consistent with the idea that protection against P. falciparum malaria in children depends on acquisition of a constellation of appropriate, functionally active IgG subclass responses directed to multiple asexual stage antigens. Our results suggest at least two distinct mechanisms via which antibodies may exert protective effects. Although declining with age, the growth inhibitory effects of purified IgG measurable in vitro reflected levels of anti-AMA1, -MSP1 and -MSP2, but not of anti-GLURP IgG. The latter could act on parasite growth via indirect parasiticidal pathways. [ABSTRACT FROM AUTHOR]

Background: Continuous high global tuberculosis (TB) mortality rates and variable vaccine efficacy of Mycobacterium bovis Bacille Calmette-Guérin (BCG) motivate the search for better vaccine regimes. Relevant models are required to downselect the most promising vaccines entering clinical efficacy testing and to identify correlates of protection. Methods and Findings: Here, we evaluated immunogenicity and protection against Mycobacterium tuberculosis in rhesus monkeys with two novel strategies: BCG boosted by modified vaccinia virus Ankara expressing antigen 85A (MVA.85A), and attenuated M. tuberculosis with a disrupted phoP gene (SO2) as a single-dose vaccine. Both strategies were well tolerated, and immunogenic as evidenced by induction of specific IFNγ responses. Antigen 85A-specific IFNγ secretion was specifically increased by MVA.85A boosting. Importantly, both MVA.85A and SO2 treatment significantly reduced pathology and chest X-ray scores upon infectious challenge with M. tuberculosis Erdman strain. MVA.85A and SO2 treatment also showed reduced average lung bacterial counts (1.0 and 1.2 log respectively, compared with 0.4 log for BCG) and significant protective effect by reduction in C-reactive protein levels, body weight loss, and decrease of erythrocyte-associated hematologic parameters (MCV, MCH, Hb, Ht) as markers of inflammatory infection, all relative to non-vaccinated controls. Lymphocyte stimulation revealed Ag85A-induced IFNc levels post-infection as the strongest immunocorrelate for protection (spearman's rho: 20.60). Conclusions: Both the BCG/MVA.85A prime-boost regime and the novel live attenuated, phoP deficient TB vaccine candidate SO2 showed significant protective efficacy by various parameters in rhesus macaques. Considering the phylogenetic relationship between macaque and man and the similarity in manifestations of TB disease, these data support further development of these primary and combination TB vaccine candidates. [ABSTRACT FROM AUTHOR]

Chimpanzees experienced a reduction of the allelic repertoire at the major histocompatibility complex (MHC) class I A and B loci, which may have been caused by a retrovirus belonging to the simian immunodeficiency virus (SIV) family. Extended MHC haplotypes were defined in a pedigreed chimpanzee colony. Comparison of genetic variation at microsatellite markers mapping inside and outside the Mhc region was carried out in humans and chimpanzees to investigate the genomic extent of the repertoire reduction. Multilocus demographic analyses underscored that chimpanzees indeed experienced a selective sweep that mainly targeted the chromosomal segment carrying the Mhc class I region. Probably due to genetic linkage, the sweep also affected other polymorphic loci, mapping in the close vicinity of the Mhc class I region genes. Nevertheless, although the allelic repertoire at particular Mhc class I and II loci appears to be limited, naturally occurring recombination events allowed the establishment of haplotype diversity after the sweep. However, recombination did not have sufficient time to erase the signal of the selective sweep. [ABSTRACT FROM AUTHOR]

Abstract: Apical membrane antigen 1 (AMA1) is a membrane protein present in Plasmodium species and is probably common to all apicomplexan parasites. The recent crystal structure of the complete ectoplasmic region of AMA1 from Plasmodium vivax has shown that it comprises three structural domains and that the first two domains are based on the PAN folding motif. Here, we discuss the consequences of this analysis for the three-dimensional structure of AMA1 from other Plasmodium species and other apicomplexan parasites, and for the Plasmodium paralogue MAEBL. Many polar and apolar interactions observed in the PvAMA1 crystal structure are made by residues that are invariant or highly conserved throughout all Plasmodium orthologues; a subgroup of these residues is also present in other apicomplexan orthologues and in MAEBL. These interactions presumably play a key role in defining the protein fold. Previous studies have shown that the ectoplasmic region of AMA1 must be cleaved from the parasite surface for host-cell invasion to proceed. The cleavage site in the crystal structure is not readily accessible to proteases and we discuss possible consequences of this observation. The three-dimensional distribution of polymorphic sites in PfAMA1 shows that these are all on the surface and that their positions are significantly biased to one side of the ectoplasmic region. Of particular note, a flexible segment in domain II, comprising about 40 residues and devoid of polymorphism, carries an epitope recognized by an invasion-inhibitory monoclonal antibody and a T-cell epitope implicated in the human immune response to AMA1. [Copyright &y& Elsevier]

Apical membrane antigen 1 from Plasmodium is a leading malariavaccine candidate. The protein is essential for host-cell invasion, butits molecular function is unknown. The crystal structure of the threedomains comprising the ectoplasmic region of the antigen from P. vivax,solved at 1.8 angstrom resolution, shows that domains I and II belong tothe PAN motif, which defines a superfamily of protein folds implicatedin receptor binding. We also mapped the epitope of an invasioninhibitorymonoclonal antibody specific for the P. falciparum ortholog and modeledthis to the structure. The location of the epitope and current knowledgeon structure-function correlations for PAN domains together suggest areceptorbinding role during invasion in which domain II plays a criticalpart. These results are likely to aid vaccine and drug design. [ABSTRACT FROM AUTHOR]

Interleukin-1β (IL-1β) is present in multiple sclerosis (MS) lesions. Interleukin-1 receptor antagonist (IL-1Ra) moderates the induction of experimental autoimmune encephalomyelitis (EAE). Here, we show that families that are characterized by high IL-1β over IL-1Ra production ratio are at 2.2-fold (95% CI, 1.0–4.8; p=0.05) increased risk to have a patient relative with relapse-onset MS than families with a low ratio. It is also related to the reduction of volumetric magnetization transfer ratio (MTR) histogram height, a measure of parenchymal integrity (p=0.04). Those families who combine a high IL-1β over IL-1Ra ratio with a high tumor necrosis factor (TNF) over IL-10 production ratio have a 6.2-fold (95% CI, 1.8–21; p=0.002) increased risk. Innate production of IL-1β and IL-1Ra is not related to the outcome of primary progressive MS. Taq1 polymorphism in the IL-1β gene and the variable number of tandem repeats (VNTR) polymorphism of 86-base pairs within the IL-1Ra gene cannot explain these findings. [Copyright &y& Elsevier]